2015
8/21
Liquid Culture
Li
Name | medium |
strain | Medium/ml |
8/20 transformation RBS | PG(AMP) /6 |
8/20 dilution INP7 | PG(CP) /6 |
8/17 transformation 3c5, RFP | PG(CP) /6 |
8/20 transformation p+ [1] | PG(AMP) /6 |
8/20 transformation p- [1] | PG(AMP) /6 |
8/21
Electrophoresis
Li
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /1.5 | |
2 | 8/21 cloning PCR scfv[1] /2 | |
3 | 8/21 cloning PCR scfv[2] /2 | |
4 | nega /2 | |
5 | 8/21 colony PCR p+ [1] /2 | |
6 | 8/21 colony PCR p+ [2] /2 | |
7 | 8/21 colony PCR p+ [3] /2 | |
8 | 8/21 colony PCR p- [1] /2 | |
9 | 8/21 colony PCR p- [2] /2 | |
10 | 8/21 colony PCR p- [3] /2 | |
11 | 8/21 colony PCR parts p+ /2 | |
12 | 8/21 colony PCR parts p- /2 | |
[img35]
2, 3, 4 cloning PCR failed.
1 forgot to put ladder
5, 7, 8, 9 strong band
6 weak
10 failed
P+[1] and p-[1] -> liquid culture
8/24
Ligation
Li
sample/µl | MilliQ (µl) | Buffer /µl | Ligase(µl) | Total(µl) |
8/19 mutation PCR scfv /5 | 3.5 | 1 | 0.5 | 10 |
8/24
Liquid Culture
Li
Name | medium |
8/20 transformation m.scfv colonyA /6 | PG(AMP) |
8/24
Miniprep
Li
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/20 Liquid culture RBS | 124.5 | 1.65 | 1.02 |
8/20 Liquid culture INP | 147.8 | 1.66 | 0.98 |
8/20 Liquid culture 3c5.RFP | 234.8 | 1.79 | 1.62 |
8/20 Liquid culture P+[1] | 40.3 | 1.78 | 1.60 |
8/20 Liquid culture P-[1] | 56.7 | 1.80 | 2.05 |
8/25
Miniprep
Li, Uchino, Notsu, Kaneko
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/24 LIquid Culture m.scfv colonyA | - | - | - |
8/24 LIquid Culture m.scfv colonyB | - | - | |
8/25
Electrophoresis
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /1.5 | |
2 | 8/24 miniprep p+(J61002) /2 | |
3 | 8/24 miniprep p-(J61002) /2 | |
4 | 8/24 miniprep RFP (3c5) /2 | |
5 | 8/24 miniprep INP (1c3) /2 | |
6 | 8/24 miniprep RBS (1A2) /2 | |
[img38]
8/25
Restriction Enzyme Digestion
Uchino
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
| p+/24.8 | | | 1 | 1 | 10xH /4 | | 9.2 | 40 |
| IINP /6.8 | | 1 | | 1 | 10xM /4 | | 27.2 | 40 |
| p- /17.6 | | | 1 | 1 | 10xH /4 | | 16.4 | 40 |
| RBS /8.0 | | | 1 | 1 | 10xH /4 | | 26 | 40 |
| RFP /4.3 | 1 | | | 1 | 10xH /4 | | 29.7 | 40 |
8/25
Gel Extraction
Uchino, Notsu
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 6 |
2, 3 | 8/25 digestion p+ | 24 each |
4, 5 | 8/25 digestion p- | 24 each |
6, 7 | 8/25 digestion RFP | 24 each |
8, 9 | 8/25 digestion INP | 24 each |
10, 11 | 8/25 digestion RBS | 24 each |
[img40]
8/25
Actvation check of Xba and Pst
Uchino
INP(µl) | Xba(µl) | Pst(µl) | 10xM(µl) | BSA(µl) | MilliQ(µl) | Total(µl) |
0.68 | 0.1 | 0.1 | 1 | 0.2 | 7.92 | 10 |
0.68 | 0.1 | - | 1 | 0.2 | 8.02 | 10 |
0.68 | - | 0.1 | 1 | 0.2 | 8.02 | 10 |
0.68 | - | - | 1 | 0.2 | 8.12 | 10 |
8/25
Electrophoresis
Notsu
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /3 | |
2 | [1] (inp xp cut) /10 | |
3 | [2] (inp x cut) /10 | |
4 | [3] (inp p cut) /10 | |
5 | [4] (inp no cut) /10 | |
[img42]
8/26
Restriction Enzyme Digestion
Uchino, Notsu
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
| 8/24 miniprep INP 1c3 /0.68 | - | 1.0 | | | | 0.2 | 7.12 | 10 |
| 8/15 miniprep RFP psb1c3 /1.3 | - | 1.0 | | | | 0.2 | 6.5 | 10 |
| 8/24 miniprep INP 1c3 /0.68 | 0.1 | 1.0 | | | | 0.2 | 7.02 | 10 |
| 8/15 miniprep RFP psb1c3 /1.3 | 0.1 | 1.0 | | | | 0.2 | 6.4 | 10 |
| 8/24 miniprep INP 1c3/ 0.68 | 0.1 | - | | | | 0.2 | 8.02 | 10 |
| 8/15 miniprep RFP psb1c3 /1.3 | 0.1 | - | | | | 0.2 | 7.4 | 10 |
| 8/24 miniprep INP 1c3 /0.68 | - | - | | | | 0.2 | 8.12 | 10 |
| 8/15 miniprep RFP psb1c3 /1.3 | - | - | | | | 0.2 | 7.5 | 10 |
8/26
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
scfv from MK | c.scfv fw/0.75 | c.scfv rv/0.75 | 2x KAPA Hifi /12.5 | 11 | | 25 |
none | c.scfv fw/0.75 | c.scfv rv/0.75 | 2x KAPA Hifi /5 | 4.4 | | 10 |
8/26
Activation Check of Xba
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/2 | |
2 | [8] RFP/10 | |
3 | [6] RFP EcoRI/10 | |
4 | [2] RFP XbaI/10 | |
5 | [4] RFP EcoRI, XbaI /10 | |
6 | [7] INP /10 | |
7 | [5] INP EcoRI /10 | |
8 | [1] INP XbaI /10 | |
9 | [3] INP EcoRI, XbaI/10 | |
[img45-1]
[img45-2]
8/27
Electrophoresis
Li
Lane | sample/(µl) | Length(bp) |
2 | 1kb ladder | 2 |
3 | 8/25 PCR scfv | 5 |
4 | 8/25 PCR none | 5 |
8/27
PCR (Steps)
Li
Steps | Temparature | Time | Cycle |
Predenature | 95 | 3min | 1 |
Denature | 98 | 20sec | 25 |
Annealing | 65 | 15sec | 25 |
Extension | 72 | 30sec | 25 |
Final Elogation | 72 | 5min | 1 |
Final Holding | 4 | - | |
8/27
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
s.BAN | BAN.Fw/ 0.75 | BAN.Rv/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | 25 |
s.BAN | BAN.Fw/ 0.75 | BAN.Rv/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | 25 |
s.BAN | BAN.Fw/ 0.75 | BAN.Histag/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | 25 |
scfv | scfv.Fw/ 0.75 | scfv.Rv/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | 25 |
8/24 miniprep INP | INP.Fw/ 0.75 | INP.Rv/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | 25 |
8/24 miniprep INP | INP.Fw/ 0.75 | INP.Rv.Histag/ 0.75 | | 11 | 2xKAPA HiFi/ 12.5 | |
8/14
Transformation
Li, Uchino, Kaneko
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
RFP 0.5ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 5ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 10ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 20ng/µl | 2 | DH5α /20 | LB(CP) |
RFP 50ng/µl | 2 | DH5α /20 | |
8/14
Gel Extraction
Li, Uchino, Kaneko
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 10/1 PQ(3) | about 23.5µl |
2 | 1kb ladder | 6 |
3 | 10/1 PQ(3) | about 23.5µl |
4 | 10/1 PQ(3) | about 24µl |
Total volume of samples of lane 1~3 was 71.5µl and it was divided into three.
8/14
Transformation
Li, Uchino, Kaneko
Sample Name | Sample Volume | Competent Cells/(µl) | Medium |
3C5 | 10µl | DH5留 20µl | LB(CP) |
P+ | 10µl | DH5留 20µl | LB(CP) |
P- | 10µl | DH5留 20µl | LB(CP) |
RBS | 10µl | DH5留 20µl | |
8/14
Liquid Culture
Li, Uchino, Iguchi, Kaneko
Name | medium |
8/13 transformation RFP 20ng/µl colony1 | LB(CP) |
8/13 transformation part RFP colony1 | LB(CP) |
8/13 transformation part RFP colony2 | LB(CP) |
8/13 transformation part RFP colony3 | LB(CP) |
8/13 transformation part RFP colony4 | LB(CP) |
8/13 transformation part RFP colony5 | LB(CP) |
8/13 transformation part RFP colony6 | LB(CP) |
8/13 transformation part RFP colony7 | LB(CP) |
8/13 transformation part RFP colony8 | LB(CP) |
8/13 transformation part RFP colony9 | LB(CP) |
8/13 transformation part RFP colony10 | LB(CP) |
| LB(CP) |
8/15
Miniprep
Li, Uchino, Notsu, Kaneko, Watanabe
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
RFP1 /2µl | 82.5 | 1.40 | 1.22 |
RFP2 /4µl | 142.5 | 1.69 | 2.05 |
RFP3 /4µl | 50 | 1.55 | 1.71 |
RFP4 /4µl | 57.5 | 1.64 | 1.88 |
RFP6 /4µl | 98.75 | 1.71 | 1.40 |
RFP(20) /4µl | 76.25 | 1.67 | |
8/15
Electrophoresis
Notsu, Kaneko
Lane | Restriction Enzyme Digestion Products |
1 | 1kb ladder 3µl |
2 | RFP1 10µl |
3 | RFP2 10µl |
4 | RFP3 10µl |
5 | RFP4 10µl |
6 | RFP6 10µl |
7 | RFP(20) 10µl |
8 | PCR1 5µl |
9 | PCR2 5µl |
10 | PCR3 5µl |
11 | PCR4 5µl |
12 | PCR5 5 µl |
13 | 1kb ladder 3µl |
8/15
Restriction Enzyme Digestion
Uchino, Notsu, Kaneko
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/15Miniprep PSB1C3-RFP1 | 24 | 1 | 0 | 0 | 1 | 5(H) | 0 | 19 | 50 |
8/15Miniprep PSB1C3-RFP2 | 14 | 1 | 0 | 0 | 1 | 5(H) | 0 | 29 | 50 |
8/15Miniprep PSB1C3-RFP3 | 40 | 1 | 0 | 0 | 1 | 5(H) | 0 | 3 | 50 |
8/15Miniprep PSB1C3-RFP4 | 35 | 1 | 0 | 0 | 1 | 5(H) | 0 | 8 | 50 |
8/15Miniprep PSB1C3-RFP6 | 20 | 1 | 0 | 0 | 1 | 5(H) | 0 | 23 | 50 |
8/15
colony PCR
Uchino, Notsu, Kaneko, Watanabe
colony Primers |
8/14 transformation pSB1C3-RFP1 VF+VR |
8/14 transformation pSB1C3-RFP2 VF+VR |
8/14 transformation pSB1C3-RFP3 VF+VR |
8/14 transformation pSB1C3-RFP4 VF+VR |
8/14 transformation pSB1C3-RFP1 VF+VR |
8/15
PCR (Steps)
Uchino, Notsu, Kaneko, Watanabe
Steps | Temparature | Time | Cycle |
Pre Denature | 94℃ | 2min | 1 |
Denature | 94℃ | 30sec | 30 |
Annealing | 55℃ | 30sec | 30 |
Extension | 68℃ | 1min | 30 |
Final elongation | 4℃ | 7min | |
Final Holding | 4℃ | - | - |
8/16
Gel Extraction
Li, Iguchi, Notsu, Kaneko, Watanabe
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 10 |
2 | blank | |
3 | 8/15 digestion pSB1C3-RFP 1 | 55 |
4 | 8/15 digestion pSB1C3-RFP 2 | 55 |
5 | 8/15 digestion pSB1C3-RFP 3 | 36 |
6 | 8/15 digestion pSB1C3-RFP 4 | 36 |
7 | 8/15 digestion pSB1C3-RFP 6 | |
8/16
Electrophoresis
Li, Iguchi, Notsu, Kaneko, Watanabe
Lane | sample/(µl) | Length(bp) |
1 | 8/16 Gel Extraction RFP1 /1 | |
2 | 8/16 Gel Extraction RFP2 /1 | |
3 | 8/16 Gel Extraction RFP3 /1 | |
4 | 8/16 Gel Extraction RFP4 /1 | |
5 | blank | |
6 | 8/16 pSB1C3-vector /1 | |
7 | 8/16 pSB1C3-vector /1 | |
8 | | |
8/16
Ligation
Li, Iguchi, Notsu, Kaneko, Watanabe
Vector DNA /(µl) insert DNA /(µl) ligase /(µl) 10×buffer /(µl) MilliQ /(µl) Total /(µl) |
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5 |
pSB1C3 EPcut /1 RFP /2 0.5 1 5 9.5 |
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5 |
pSB1C3 EPcut /1 RFP /4 0.5 1 3 9.5 |
pSB1C3 EPcut /1 - 0.5 1 7 9.5 |
8/17
Liquid Culture
Li
Name | medium |
8/16 transformation pSB3C5 RFP1 | LB(CP) /6 |
8/16 transformation pSB3C5 RFP2 | LB(CP) /6 |
8/16 transformation pSB3C5 RFP1 | LB(CP) /6 |
8/17
Transformation
Li
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
pSB1C3 P+ | 2 | DH5α /20 | LB |
pSB1C3 P- | 2 | DH5α /20 | LB |
pSB1C3 RBS | 2 | DH5α /20 | LB |
pSB3C5 RFP | 2 | DH5α /20 | LB |
8/16 ligation pSB1C3 RFP1 | 2 | DH5α | LB |
8/17
Electrophoresis
Iguchi
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder /3 | |
2 | blank | |
3 | pSB1C3-RFP(ligate)1 /2 | |
4 | pSB1C3-RFP(ligate)2 /2 | |
5 | pSB1C3-RFP(ligate)3 /2 | |
6 | pSB1C3-RFP(ligate)4 /3 | |
7 | Vector (Ctrl) /5 | |
8/18
Liquid Culture
Li, Iguchi, Notsu
Name | medium |
INP [1] BBa_K811005 | LB(CP) / 6ml |
INP [2] BBa_K811005 | LB(CP) / 6ml |
INP [3] BBa_K811005 | LB(CP) / 6ml |
scfv04 [1] BBa_K875004 | LB(CP) / 6ml |
scfv04 [2] BBa_K875004 | LB(CP) / 6ml |
scfv04 [3] BBa_K875004 | LB(CP) / 6ml |
scfv06 [1] BBa__K875006 | LB(CP) / 6ml |
scfv06 [2] BBa_K875006 | LB(CP) / 6ml |
scfv06 [3] BBa_K875006 | LB(CP) / 6ml |
8/18
Miniprep
Li, Iguchi, Notsu
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/17 Liquid Culture 3C5 [1] / 5µl | 77 | 1.69 | 1.45 |
8/17 Liquid Culture 3C5 [2] / 2µl | 40 | 1.67 | 1.17 |
8/17 Liquid Culture 3C5 [3] / 2µl | 15 | 3.75 | 3.70 |
8/18
Electrophoresis
Li
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /1 | - |
2 | [1] psb3c5(8/18 miniprep) /4.2 | 3807 |
3 | [2] psb3c5(8/18 miniprep) /4.2 | 3807 |
4 | [1'] psb3c5(8/18 miniprep) /4.2 | 3807 |
5 | [2'] psb3c5(8/18 miniprep) /4.2 | 3807 |
6 | [3'] psb3c5(8/18 miniprep) /4.2 | 3807 |
![]()
https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--8_18_No18_Electrophoresis_Kaneko_05_20_04_12_53.jpg
[img18]
8/18
Restriction Enzyme Digestion
Li, Iguchi, Notsu
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
| psb3c5(8/18miniprep) [1'], [2'], [3'] mixed / 50 | 1.0 | | | 1.0 | 5.6 | | | 56.6 |
8/19
Transformation
Li, Iguchi, Notsu
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
BBa_ J61002 p+ | 2 | DH5a /20 | LB(Amp) |
BBa_J61002 p- | 2 | DH5a /20 | LB(Amp) |
BBa_1A2 RBS | 2 | DH5a /20 | LB(Amp) |
Failed due to deactivation of Amp
8/19
MutationPCR
Templates /µl | Primer /µl | Primer STAR Max Premix /µl | MilliQ /µl | Total(µl) |
scfv plasmid /0.1 | m.scfv FW /1, m.scfvRV/1 | 25 | 22.9 | 50 |
scfv plasmid (nega) /0.1 | 0 | 5 | 4.9 | 10 |
(nega) /0 | m.scfv FW/0.2, m.scfvRV/0.2 | 5 | 4.6 | 10 |
---30cycle---
98℃ 10''
55℃ 15''
72℃ 20''
----------------
4℃ holding
8/19
Electrophoresis
Notsu
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/2 | - |
2 | scfv plasmid +primer(m.scfv FW, m.scfv RV) mutation PCR product /5 | 3600 |
3 | scfv plasmid mutation PCR product /5 | - |
4 | primer(m.scfv FW, m.scfv RV) mutation PCR product /5 | - |
5 | psb3c5 EPcut product /5 | 2600(backborn), 1200(inseert) |
[img22]
8/19
Concentration
Li, Notsu
Sample | OD600 | Bacterial cells /ml |
INP [1] K811005 | 0.643 | 5.14x10^8 |
scfv06 K875006 | 0.634 | 5.07x10^8 |
scfv04 K875004 | 0.618 | 4.94x10^8 |
1 OD 600 equals to 8x10^8 cells /ml
8/19
Miniprep
Li, Notsu
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
scfv 04[1] K875004 / 3µl | 56 | 1.63 | 1.45 |
scfv 04[2] K875004 / 3µl | | | |
scfv 04[3] K875004 / 3µl | | | |
scfv 06[1] K875006 /3µl | | | |
scfv 06[2] K875006 /3µl | | | |
scfv 06[3] K875006 /3µl | | | |
INP [1] K811005 /3µl | | | |
INP [2] K811005 /3µl | | | |
INP [3] K811005 /3µl | | | |
There was a problem with an instrument
8/20
Transformation
Li, Uchino
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/19 mutation PCR scfv plasmid (nega) | 1 | DH5a/ 20 | LB(CP) |
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.
8/20
Transformation
Li, Uchino
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/19 mutation PCR scfv plasmid | 1 [note:1] | DH5a/20 | LB(AMP) |
8/19 mutation PCR scfv plasmid (nega) | 1 [note:2] | DH5a/20 | LB(AMP) |
BBa_J61002 p+ | 1 | DH5a/20 | LB(AMP) |
BBa_J61002 p- | 1 | DH5a/20 | LB(AMP) |
BBa_1A2 RBS | 1 | DH5a/20 | LB(AMP) |
[note:1]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/50µl was folded 300 times.
[note:2]
8/19 mutation PCR:
1µl extraction of scfv plasmid (nega)/10µl was folded 300 times.
8/20
Electrophoresis
Li
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /1.5 | |
2 | 9/19 miniprep scfv04 [1] | |
3 | 9/19 miniprep scfv04 [2] | |
4 | 9/19 miniprep scfv04 [3] | |
5 | 9/19 miniprep scfv06 [1] | |
6 | 9/19 miniprep scfv06 [2] | |
7 | 9/19 miniprep scfv06 [3] | |
8 | 9/19 miniprep INP [1] | |
9 | 9/19 miniprep INP [2] | |
10 | 9/19 miniprep INP [3] | |
[img26]
8/21
Colony PCR
Uchino
Colony | Primers |
8/20 transformation RBS | VF+VR |
8/20 transformation p+ | VF+VR |
8/20 transformation p- | VF+VR |
8/20 dilution INP | VF+VR |
8/21
PCR (Steps)
Uchino
Steps | Temparature | Time | Cycle |
Pre | 94℃ | 2min | 1 |
De | 94℃ | 30 sec | 30 |
An | 55℃ | 30 sec | 30 |
Ex | 68℃ | 1min | 30 |
Final E | 4℃ | 7min | 1 |
Final Hold | 4℃ | - | - |
Autoclaved distrilled water /23µl
2xQuick taq /25µl
10 pmol/µl primer (VF) /1µl
10 pmol/µl primer (VR) /1µl
Template DNA /0µl
Total 50µl
8/21
Electrophoresis
Li
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /2 | |
2 | 8/21 colony PCR RBS /all | |
3 | 8/21 colony PCR p+ /all | |
4 | 8/21 colony PCR p- /all | |
5 | 8/21 colony PCR INP /all | |
6 | 8/21 colony PCR Nega /all | |
![](https://static.igem.org/mediawiki/2016/0/04/T--Kyoto--8_21_No28_Electrophoresis_Kaneko_05_20_04_54_01%2C1.jpg)
[img29-1]
weak band except INP -> retry
colony PCR and Electrophoresis were conducted under the same condition.
[img29-2]
RBS and INP success.
P+ and P- failed -> retry.
8/21
PCR (Steps)
Li
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 25 |
Annealing | 70℃ | 15sec | 25 |
Extension | 72℃ | 30sec | 25 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | 1 |
8/21
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
scfv 8/20 transformation | cscfv.f /0.75 | cscfv.r /0.75 | 2x KAPA Hifi /12.5 | 11 | | 25 |
- (nega) | cscfv.f /0.3 | cscfv.f /0.3 | 2x KAPA Hifi /5 | 4.4 | | 10 |
8/21
Colony PCR
Li
Colony | Primers |
8/20 transformation p+ [1] | VF+VR |
8/20 transformation p+ [2] | VF+VR |
8/20 transformation p+ [3] | VF+VR |
8/20 transformation p- [1] | VF+VR |
8/20 transformation p- [2] | VF+VR |
8/20 transformation p- [3] | VF+VR |
8/18 parts p+ | VF+VR |
8/18 parts p- | VF+VR |
8/21
PCR (Steps)
Li
Steps | Temparature | Time | Cycle |
pre | 94℃ | 2min | 1 |
de | 94℃ | 30sec | 30 |
an | 55℃ | 30sec | 30 |
ex | 68℃ | 1min | 30 |
F.elong | 4℃ | 7min | 1 |
F.hold | 4℃ | - | 1 |
9/8
Restriction Enzyme Digestion
Notsu
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
| [1] 8/15 miniprep 1c3 RFP /14 | 0.5 | | 0.5 | - | 10xH /3 | | 12 | 30 |
| [2] 8/24 miniprep 3c5 RFP /8.5 | 0.5 | | - | 0.5 | 10xH /3 | | 17.5 | 30 |
| [3] 8/24 miniprep 3c5 RFP /8.5 | 0.5 | | 0.5 | - | 10xH /3 | | 17.5 | 30 |
| [4] 8/24 miniprep BBa_J61002 p- /24 | 0.5 | | - | 0.5 | 10xH /3 | | 2 | 30 |
9/9
Electrophoresis
Notsu
Lane | Restriction Enzyme Digestion Products |
1 | 100bp ladder 9µL |
2 | 8/24 miniprep p- EPcut 30µL |
9/9
Electrophoresis
Sukegawa, Notsu
Lane | Restriction Enzyme Digestion Products |
1 | 1kbp ladder 9µL |
2 | 8/15 miniprep 1C3RFP EScut 30µL |
3 | 8/24 miniprep 3C5RFP EPcut 30µL |
4 | 8/24 miniprep 3C5RFP EScut 30µL |
9/14
PCR (Steps)
Li
Steps | Temparature | Time | Cycle |
1 | 95 | 3min | 1 |
2 | 98 | 70sec | 25 |
3 | 68 | 15sec | 25 |
4 | 72 | 30sec | 25 |
5 | 72 | 5min | 1 |
6 | 10 | ∞ | 1 |
9/14
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
8/24 miniprep INP | INPfw/0.75 | INPRv/0.75 | 2×KAPAHiFi | 11 | | 25 |
8/24 miniprep INP | INPfw/0.75 | INPHisRv/0.75 | 2×KAPAHiFi | 11 | | 25 |
BAN | BANFw/0/75 | BANRv/0.75 | 2×KAPAHiFi | 11 | | 25 |
BAN | BANFw/0/75 | BANHisRv/0.75 | 2×KAPAHiFi | 11 | | 25 |
BAN | BANFw/0/75 | RBSRv/0.75 | 2×KAPAHiFi | 11 | | 25 |
scFv fromMk | scFvFw/0/75 | scFvRv/0/75 | 2×KAPAHiFi | 11 | | 25 |
scFv fromMk | scFvHisFw/0.75 | | 2×KAPAHiFi | 11 | | 25 |
9/14
Gel Extraction
Kaneko
Lane | Restriction Enzyme Digestion Products |
2 | ladder/6 |
4 | 9/14 PCR1/10 |
6 | 9/14 PCR2/10 |
8 | 9/14 PCR3/10 |
10 | 9/14 PCR4/10 |
12 | 9/14PCR5/10 |
14 | 9/14 PCR6/10 |
16 | 9/14 PCR7/10 |
![](https://static.igem.org/mediawiki/2016/4/4b/T--Kyoto--9_14_No53_Electrophoresis_Yamamoto_08_23_11_27_01.1.JPG)
9/14
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3sBAN/0.15+6scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | | | 25 |
4sBAN/0.15+7scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | | | 25 |
1INP8/24miniprep/0.15+5sBAN0.15+scFvfromMK/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | | | 25 |
2INP8/24miniprep/0.15 | BANFw/0/75 | scFvRv/0.75 | 2×KAPAHiFi/12.5 | | | 25 |
9/15
Restriction Enzyme Digestion
Li, Uchino
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3sBAN+6scFvfromMKPCRproduct | 34 | 1 | | 1 | | 4 | | | 40 |
4sBAN+7scFvfromMK PCRproduct | 34 | 1 | | 1 | | 4 | | | 40 |
1INP8/24miniprep+5sBAN+6scFvfromMKPCRproduct | 16 | 1 | | 1 | | 2 | | | 20 |
2INP8/24miniprep+5sBAN+7scFvfromMk PCRproduct | | 1 | | 1 | | 4 | | | 40 |
9/15
Gel Extraction
Li, Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | |
2 | 3sBAN+6scFvfromMkPCR産物EScut | 20 |
3 | 3sBAN+6scFvfromMkPCR産物EScut | 20 |
4 | 4sBAN+7scFvfromMkPCR産物EScut | 20 |
5 | 4sBAN+7scFvfromMkPCR産物EScut | 20 |
6 | 1INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut | 20 |
7 | 2INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut | 20 |
8 | 2INP8/24miniprep+5sBAN+7scFvfromMkPCR産物EScut | 20 |
9 | RFP3C5PCR産物 | |
![](https://static.igem.org/mediawiki/2016/a/a2/T--Kyoto--9_15_No56_Gel_Extraction_Yamamoto_08_23_11_46_18%2C1.jpg)
9/16
Colony PCR (2Column)
Uchino, Michimori
Name I | Name II |
9/15 transformation BANscFv 3C5 colony1~16 | VF+VR |
9/15 transformation INPscFv 3C5 colony1~16 | VF+VR |
9/15 transformation BANHisscFv 3C5 colony1~16 | VF+VR |
9/15 transformation INPHisscFv 3C5 colony1~16 | |
9/16
PCR (Steps)
Uchino, Michimori
Steps | Temparature | Time | Cycle |
1 | 94 | 2 | 1 |
2 | 94 | 0.5 | 25 |
3 | 55 | 0.5 | 25 |
4 | 68 | 1 | 25 |
5 | 4 | 7 | 1 |
6 | 4 | ∞ | |
9/16
Gel Extraction
Li, Kaneko, Notsu
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | BANscFv3C5 colony 9 | 10 |
3 | BANscFv3C5 colony 10 | 10 |
4 | BANscFv3C5 colony 11 | 10 |
5 | BANscFv3C5 colony 12 | 10 |
6 | BANscFv3C5 colony 13 | 10 |
7 | BANscFv3C5 colony 14 | 10 |
8 | BANscFv3C5 colony 15 | 10 |
9 | BANscFv3C5 colony 16 | 10 |
10 | BANHisscFv3C5 colony 9 | 10 |
11 | BANHisscFv3C5 colony 10 | 10 |
12 | BANHisscFv3C5 colony 11 | 10 |
13 | BANHisscFv3C5 colony 12 | 10 |
14 | BANHisscFv3C5 colony 13 | 10 |
15 | BANHisscFv3C5 colony 14 | 10 |
16 | BANHisscFv3C5 colony 15 | 10 |
17 | BANHisscFv3C5 colony 16 | 10 |
18 | BANscFv3C5 colony 1 | 10 |
![](https://static.igem.org/mediawiki/2016/d/d3/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.1.JPG)
![](https://static.igem.org/mediawiki/2016/3/34/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.2.JPG)
![](https://static.igem.org/mediawiki/2016/a/aa/T--Kyoto--9_16_No59_Gel_Extraction_Yamamoto_08_23_11_57_19.3.JPG)
9/16
Gel Extraction
Li, Kaneko, Notsu
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | INPscFv3C5 colony 9 | 10 |
3 | INPscFv3C5 colony 10 | 10 |
4 | INPscFv3C5 colony 11 | 10 |
5 | INPscFv3C5 colony 12 | 10 |
6 | INPscFv3C5 colony 13 | 10 |
7 | INPscFv3C5 colony 14 | 10 |
8 | INPscFv3C5 colony 15 | 10 |
9 | INPscFv3C5 colony 16 | 10 |
10 | INPHisscFv3C5 colony 9 | 10 |
11 | INPHisscFv3C5 colony 10 | 10 |
12 | INPHisscFv3C5 colony 11 | 10 |
13 | INPHisscFv3C5 colony 12 | 10 |
14 | INPHisscFv3C5 colony 13 | 10 |
15 | INPHisscFv3C5 colony 14 | 10 |
16 | INPHisscFv3C5 colony 15 | 10 |
17 | INPHisscFv3C5 colony 16 | 10 |
18 | BANscFv3C5 colony 1 | 10 |
![](https://static.igem.org/mediawiki/2016/3/3d/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C1.jpg)
![](https://static.igem.org/mediawiki/2016/8/88/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C2.jpg)
![]()
![](https://static.igem.org/mediawiki/2016/4/42/T--Kyoto--9_16_No60_Gel_Extraction_Yamamoto_08_23_11_59_27%2C3.jpg)
9/17
Electrophoresis
Michimori, Kaneko
Lane | Restriction Enzyme Digestion Products |
1 | buffer/5 |
2 | 1kb ladder/5 |
3 | BAN 9 P/4.5 |
4 | BAN 9 N/4.5 |
5 | BAN14 P/4.5 |
6 | BAN14 N/4.5 |
7 | BANHis14 P/4.5 |
8 | BANHis14/4.5 |
9/17
Electrophoresis
Kaneko
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder | |
2 | none | |
3 | BANHis16 p | |
4 | BANHis16 n | |
5 | INP 12 p | |
6 | INP 14 p | |
7 | INP 12 n | |
8 | | |
9/17
Electrophoresis
Kaneko
Lane | Restriction Enzyme Digestion Products |
1 | - |
2 | - |
3 | 1kbladder/5 |
4 | INPHis9 P/4.5 |
5 | INPHis9N/4.5 |
6 | INPHis12P/4.5 |
7 | INPHis12N/4.5 |
8 | INP14 N/4.5 |
9/17
Restriction Enzyme Digestion
Uchino, Michimori
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
BAN9 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
BAN9 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
BAN14 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
BAN14 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
BANHis14 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
BANHis 14 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
BANHis 16 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
BANHis16 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
INP12 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
INP12 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
INP14 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
INP 14 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
INP 9 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
INP9 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
INPHis12 | 2 | 0.5 | | | 0.5 | 10×H/1 | | 6 | 10 |
INPHis12 | 1 | 0 | | | 0 | 10×H/1 | | 8 | 10 |
9/17
Miniprep
Notsu
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
9/16 liquid culture BANscFv12 | | | |
9/16 liquid culture BANscFv14 | | | |
9/16 liquid culture BANHisscFv12 | | | |
9/16 liquid culture BANHisscFv14 | | | |
9/16 liquid culture INPHisscFv12 | | | |
9/16 liquid culture INPHisscFv14 | | | |
9/16 liquid culture INPHisscFv10 | | | |
9/16 liquid culture BANscFv | | | |
2016
2/4
PartsAwaking
Kaneko, Wan
Description | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
RFP | E1010 | pSB1C3 | 11N | 2015 | 3 | |
2/4
Transformation
Uchino, Iguchi, Notsu, Wan
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
E1010 | 2 | DH5α | LB(CP) |
2/5
Colony PCR (typeA)
Uchino, Egashira, Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/4 transformation E1010 colony1 | VF 6.25µl VR 6.25µl | 12.5 | 2×Quick Taq HS DyeMix 25µl | 50 |
2/5
PCR (Steps)
Uchino, Egashira, Yamamoto
Steps | Temparature | Time | Cycle |
Predenature | 94℃ | 2min | 1 |
Denature | 94℃ | 30sec | 25 |
Annealing | 51℃ | 30sec | 25 |
Extension | 68℃ | 90sec | 25 |
Final Elongation | 4℃ | - | - |
Final Holding | 4℃ | - | |
2/5
Transformation
Uchino
Sample Name | Sample Volume | Competent Cells/(µl) | Medium |
9/17 miniprep BAN9 | 2 | DH5α/20µL | |
9/17 miniprep BANHis14 | 2 | DH5α/20µL | |
9/17miniprep INPHis12 | 2 | DH5α/20µL | |
9/17miniprep INPHis9 | 2 | | |
2/5
Liquid Culture
Uchino, Wan, Yamamoto
Name | medium |
2/4 transformation E1010 colony1 | |
2/5
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 2/5 colony PCR E1010 | 1019 |
3 | other sample | - |
4 | other sample | - |
5 | other sample | - |
6 | other sample | - |
7 | other sample | - |
8 | other sample | |
2/6
Miniprep
Notsu, Kaneko, Egashira, Wan, Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/5 liquid culture E1010(RFP) colony1 | 48.0 | 1.93 | |
2/6
Restriction Enzyme Digestion
Kaneko, Egashira, Wan, Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/6 miniprep E1010 colony1 | 34 | 0 | 1 | 0 | 1 | 1×M /4 | 0 | 0 | 40 |
2/7
Gel Extraction
Kaneko, Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 10 |
2 | E1010 colony1 2/6 XPcut | 40 |
3 | other sample | - |
4 | other sample | - |
![](https://static.igem.org/mediawiki/2016/8/84/T--Kyoto--2_7_No78_Gel_Extraction_Notsu_10_01_04_37_25.1.JPG)
2/7
Gel Extraction(Measurement)
Kaneko, Yamamoto
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
E1010 colony1 2/6 XPcut | 8.2 | 1.03 | 0.49 |
2/7
Liquid Culture
Uchino
Name | medium |
2/5 transformation BAN9 colony1 | LB(CP) |
2/5 transformation BAN-HIS14 colony1 | LB(CP) |
2/5 transformation INP-HIS12 colony1 | LB(CP) |
2/5 transformation BAN9 colony1 | LB(CP) |
2/8
Liquid Culture
Uchino, Yamamoto
Name | medium |
2/4 transformation E1010 colony2 1 | LB(CP) |
2/4 transformation E1010 colony2 2 | LB(CP) |
2/8
Miniprep
Uchino, Egashira, Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/7 Liquid culture BAN9 | 123.6 | 1.87 | 2.07 |
2/7 Liquid culture BAN9 | 248.8 | 1.83 | 1.84 |
2/7 Liquid culture BAN-HIS14 | 268.9 | 1.86 | 2.15 |
2/7 Liquid culture BAN-HIS14 | 127.9 | 1.85 | 2.08 |
2/7 Liquid culture INP-HIS9 | 354.3 | 1.86 | 2.24 |
2/7 Liquid culture INP-HIS9 | 289.4 | 1.85 | 1.92 |
2/7 Liquid culture INP-HIS12 | 172.3 | 1.87 | 2.07 |
2/7 Liquid culture INP-HIS12 | 286.5 | 1.86 | |
2/8
Electrophoresis
Uchino, Egashira, Yamamoto
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 2/8 miniprep BAN9 /5 | |
3 | 2/8 miniprep BAN9 /5 | |
4 | 2/8 miniprep BAN-HIS14 /5 | |
5 | 2/8 miniprep BAN-HIS14 /5 | |
6 | 2/8 miniprep INP-HIS9 /5 | |
7 | 2/8 miniprep INP-HIS9 /5 | |
8 | 2/8 miniprep INP-HIS14 /5 | |
9 | 2/8 miniprep INP-HIS9 /5 | |
2/9
Miniprep
Matsumoto, Notsu, Egashira, Yamamoto, Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/8 liquid culture E1010 colony2 1and 2 | 58.5 | 1.89 | 2.16 |
2/9
Restriction Enzyme Digestion
Uchino, Egashira, Yamamoto, Yamada
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/8 liquid culture E1010 colony2 | 30 | 0 | 1 | 0 | 1 | 10×M /4 | 2 | 2 | 40 |
2/9
Restriction Enzyme Digestion
Uchino, Yamamoto, Yamada
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/8 liquid culture E1010 colony2 | 5 | 0 | 0 | 0 | 0 | 10×M /2 | 1 | 12 | 20 |
2/10
Electrophoresis
Uchino, Yamamoto, Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 2/9 Negative Control E1010 colony2 /10 | 2776 |
3 | 2/9 Digestion E1010 colony2 p+x /40 | 732 |
4 | 2/9 Negative Control E0040 colony2 /20 | 2799 |
5 | 2/9 Digestion E0040 colony2 p+x /40 | 746 |
6 | 2/9 Negative Control K592009 colony2 /20 | 2739 |
7 | 2/9 Digestion K592009 colony2 p+x /40 | 695 |
8 | 2/9 Negative Control E0022 colony2 /20 | 2832 |
9 | 2/9 Digestion E0022 colony2 p+x /40 | |
Lane7流出
2/10
Electrophoresis
Uchino, Yamamoto, Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
3 | 2/9 NC J23105 colony2 /20 | 2105 |
4 | 2/9 Digestion J23105 colony2 E+S /40 | 58 |
5 | 2/9 NC J23100 colony2 /20 | 2105 |
6 | 2/9 Digestion J23100 colony2 E+S /40 | 58 |
7 | 2/9 NC B0034 colony3 /20 | 2091 |
8 | 2/9 Digestion B0034 colony3 P+S /40 | |
![](https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.1.JPG)
![](https://static.igem.org/mediawiki/2016/4/45/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.2.JPG)
![](https://static.igem.org/mediawiki/2016/7/73/T--Kyoto--2_10_No91_Electrophoresis_Takimoto_09_14_05_48_30.3.JPG)
2には何も入れてない
2/10
Miniprep
Michimori, Egashira, Yamamoto, Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
E1010 | 9.4 | 2.13 | 0.61 |
K592009 | 5.2 | 1.74 | 0.29 |
E0040 | 15.0 | 1.94 | 0.38 |
E0022 | 8.5 | 1.04 | 0.86 |
B0034 | 21.2 | 1.84 | 1.06 |
E1010 理想値18.5ng/µLぐらい...
(制限酵素処理100%、ゲル抽出80%として)
2/12
PartsAwaking
Uchino
Description | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
sfGFP-CBDcex | K1321342 | pSB1C3 | 18E | 2015 | 5 | 10 |
sfGFP-CBDclos | K1321341 | pSB1C3 | 18C | 2015 | 5 | 10 |
2/12
Transformation
Uchino
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/12 parts awakening sfGFP-CBDcex | 1 | DH5α /20 | LB(CP) |
2/12 parts awakening sfGFP-CBDclos | 1 | DH5α /20 | LB(CP) |
2/12
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 miniprep BAN-His14 4 /0.1 | VF /0.75 | m.scFv.Rv /0.75 | - | 10.9 | 2×KAPA HiFi /12.5 | 25 |
2/8 miniprep BAN-His14 4 /0.1 | VR /0.75 | m.scFv.Fw /0.75 | - | 10.9 | 2×KAPA HiFi /12.5 | 25 |
2/12
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | 5 |
2 | 2/12 PCR VF.m.scFv.Rv | 25 |
3 | 2/12 PCR VR.m.Fw | 25 |
2/12
PartsAwaking
Uchino
Description | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
Promoter | J23114 | J61002 | 19J | 2015 | 4 | 10 |
Promoter | J23108 | pSB1C3 | 4C | 2015 | 4 | 10 |
2/12
Transformation
Uchino, Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/12 Parts awakening J23114 | 1 | DH5α /20 | LB(AMP) |
2/12 Parts awakening J23108 | 1 | DH5α /20 | LB(AMP) |
2/12
Gel Extraction(Measurement)
Yamada
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/12 PCR VF.m.scFv.Rv | 18.8 | 1.95 | 1.52 |
2/12 PCR VR.m.scFv.Fw | 28.6 | 2.08 | 0.07 |
2/15
Colony PCR (typeA)
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/12 transformation sfGFP-CBDcex colony1 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDcex colony2 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDclos colony1 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation sfGFP-CBDclos colony2 | VF /0.75 VR /0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/15
Electrophoresis
Kaneko
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 2/12 transformation sfGFP-CBDcex colony1 | |
3 | 2/12 transformation sfGFP-CBDcex colony2 | |
4 | 2/12 transformation sfGFP-CBDclos colony1 | |
5 | 2/12 transformation sfGFP-CBDclos colony2 | |
2/15
Liquid Culture
Wan, Yamamoto
Name | medium |
2/12 transformation sfGFP-CBDcex colony2 | LB(CP) |
2/12 transformation sfGFP-CBDclos colony2 | LB(CP) |
2/15
Colony PCR (typeA)
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/12 transformation J23114 colony1 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23114 colony2 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23108 colony1 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/12 transformation J23108 colony2 | VF 0.75 VR 0.75 | 11 | 2×KAPA HiFi /12.5 | 25 |
2/15
PCR (Steps)
Uchino
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/15
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35 | VF /0.75 | VR /0.75 | - | 10.12 | 2×KAPA HiFi /12.5 | 25 |
2/12 Gel extraction PCR VF/m.scFv.Rv /0.53 + 2/12 Gel extraction PCR VR/m.scFv.Fw /0.35 | VF /0.75 | VR /0.75 | - | 10.12 | 2×KAPA HiFi /12.5 | 25 |
2/15
Electrophoresis
Kaneko
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | other sample | - |
3 | other sample | - |
4 | other sample | - |
5 | other sample | - |
6 | other sample | - |
7 | other sample | - |
8 | other sample | - |
9 | other sample | - |
10 | other sample | - |
11 | other sample | - |
12 | other sample | - |
13 | other sample | - |
14 | 2/12 transformation J23114 colony1 /10 | - |
15 | 2/12 transformation J23114 colony2 /10 | |
16 | 2/12 transformation J23108 colony1 /10 | |
17 | 2/12 transformation J23108 colony2 /10 | |
2/15
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1ka ladder | 5 |
2 | 2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 25 |
3 | 2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 25 |
![](https://static.igem.org/mediawiki/2016/8/85/T--Kyoto--2_15_No114_Gel_Extraction_Notsu_10_01_08_21_26.1.JPG)
2/15
Liquid Culture
Wan, Yamada
Name | medium |
2/12 transformation J23114 colony2 | LB(AMP) |
2/12 transformation J23108 colony2 | LB(AMP) |
2/16
Miniprep
Wan, Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/15 liquid culture sfGFP-CBDcex colony2 | 43.5 | 1.91 | 1.60 |
2/15 liquid culture sfGFP-CBDclos colony2 | 74.0 | 1.86 | 1.82 |
2/16
PCR (Steps)
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/16
PCR (Target)
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/16 miniprep sfGFP-CBDcex colony2 /0.3 | cex.Fw /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16 miniprep sfGFP-CBDclos colony2 /0.3 | cex.Fw /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16
Gel Extraction(Measurement)
Yamamoto
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/15 OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw | 48.6 | 1.83 | 0.19 |
2/16
PCR (Target)
Yamamoto, Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3 | VF /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/15 Gel Extraction OE-PCR VF/m.scFv.Rv+VR/m.scFv.Fw /0.3 | VF /0.75 | VR /0.75 | - | 10.7 | 2×KAPA HiFi /12.5 | 25 |
2/16
Miniprep
Uchino, Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/15 liquid culture J23114 | 178.8 | 1.91 | 2.20 |
2/16
PCR (Purification)
Yamada
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
2/16 PCR m.scFv | 152.6 | 1.86 | 2.10 |
2/16
Restriction Enzyme Digestion
Matsumoto, Notsu, Wan
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/16 PCR m.scFv | 4 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 11 | 30 |
K592009 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
2/16
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | m.scFv 2/16 EPcut | 30 |
3 | K592009 2/16EPcut | 30 |
2/16
Liquid Culture
Uchino
Name | medium |
2/12 transformation J23108 colony3 | LB(AMP) |
2/17
PCR (Steps)
Uchino, Kim
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 25 |
Annealing | 65℃ | 15sec | 25 |
Extension | 72℃ | 30sec | 25 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/17
PCR (Target)
Uchino, Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 miniprep BAN-His14 /0.1 | BAN-His-cex.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep BAN-His14 /0.1 | BAN-His-clos.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His12 /0.1 | INP-His-cex.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His12 /0.1 | INP-His-clos.Rv /0.75 | VF /0.75 | - | 10.9 | KAPA HiFi Ready Mix /12.5 | 25 |
2/17
Gel Extraction
Michimori, Wan, Yamamoto, Kim
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | blank | |
2 | 2/17PCR BAN-His-CBDcexRv+VF | 25 |
3 | 2/17PCR BAN-His-CBDclosRv+VF | 25 |
4 | 2/17PCR INP-His-CBDcexRv+VF | 25 |
5 | 2/17PCR INP-His-CBDclosRv+VF | 25 |
6 | 2/16PCR sf-GFP-CBDcex | 50 |
7 | 2/16PCR sf-GFP-CBDclos | 50 |
8 | blank | - |
9 | 1kb ladder | - |
2/17
Gel Extraction(Measurement)
Michimori, Wan, Yamamoto, Kim
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/17PCR BAN-His-CBDcexRv+VF | 10.0 | 1.86 | 0.43 |
2/17PCR BAN-His-CBDclosRv+VF | 34.6 | 1.91 | 1.81 |
2/17PCR INP-His-CBDcexRv+VF | 100.2 | 1.90 | 1.24 |
2/17PCR INP-His-CBDclosRv+VF | 116.5 | 1.90 | 2.08 |
2/16PCR sf-GFP-CBDcex | 244.0 | 1.91 | 2.08 |
2/16PCR sf-GFP-CBDclos | 208.9 | 1.90 | 2.01 |
2/17
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 3min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 65℃ | 15sec | 35 |
Extension | 72℃ | 30sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/17
PCR (Target)
Yamamoto
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/17 Gel Extraction BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex /0.5 | VF /0.75 | VR /0.75 | - | 2 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos /2 | VF /0.75 | VR /0.75 | - | 2 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex /1 | VF /0.75 | VR /0.75 | - | 3.5 | 2×KAPA HiFi /12.5 | 25 |
2/17 Gel Extraction INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos /2 | VF /0.75 | VR /0.75 | - | 0 | 2×KAPA HiFi /12.5 | 25 |
2/17
Miniprep
Matsumoto, Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/16 liquid culture J23108 colony3 | 20.8 | 1.90 | 1.28 |
2/17
Gel Extraction(Measurement)
Notsu, Wan
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/16 Gel Extraction m.scFv EPcut | 14.3 | 1.87 | 1.09 |
2/16 Gel Extraction K592009 EPcut | 14.1 | 1.86 | 1.60 |
2/17
Ligation
Wan, Yamamoto
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
2/16 Gel Extraction K592009 EPcut /2.5 | 2/16 Gel Extraction m.scFv EPcut /5.0 | 7.5 | 15 |
2/17
Transformation
Wan, Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/17 ligation m.scFv EPcut+K592009 EPcut | 2 | DH5α /20 | LB(CP) |
2/18
Gel Extraction(Measurement)
Kim
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 72.7 | 1.87 | 1.93 |
2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos | 112.1 | 1.92 | 1.34 |
2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 102.4 | 1.89 | 1.96 |
![](https://static.igem.org/mediawiki/2016/a/ac/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_28_40.1.jpg)
2/18
Gel Extraction(Measurement)
Kim
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos | 103.4 | 1.94 | 2.04 |
![](https://static.igem.org/mediawiki/2016/9/97/T--Kyoto--2_18_No11_Gel_Extraction%28Measurement%29_Notsu_10_02_07_32_29.1.jpg)
2/18
Gel Extraction
Kim
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 8 |
2 | 2/17 OE-PCR BAN-His-CBDcexRv+VF /8.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 25 |
3 | 2/17 OE-PCR BAN-His-CBDclosRv+VF /7 + 3/17 Gel Extraction 2/16PCR sf-GFP-CBDclos | 25 |
4 | 2/17 OE-PCR INP-His-CBDcexRv+VF /6.5 + 3/17 Gel Extraction sf-GFP-CBDcex | 25 |
2/18
Gel Extraction
Kim
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 8 |
2 | 2/17 OE-PCR INP-His-CBDclosRv+VF /9 + 3/17 Gel Extraction sf-GFP-CBDclos | 25 |
3 | blank | - |
4 | blank | - |
2/18
Gel Extraction(Measurement)
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
BAN-CBDcex | 72.3 | 1.85 | 1.87 |
BAN-CBDclos | 79.8 | 1.86 | 1.26 |
INP-CBDcex | 83.6 | 1.80 | 1.57 |
INP-CBDclos | 39.4 | 1.76 | 1.77 |
2/18
PCR (Target)
Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
BAN-CBDcex /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
BAN-CBDclos /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
INP-CBDcex /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
INP-CBDclos /0.2 | VF /1.25 | VR /1.25 | - | 9.8 | KAPA Pre Mix /12.5 | 25 |
2/18
Colony PCR (typeA)
Uchino
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/17 transformation m.scFv EPcut+K592009 EPcut colony1 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony2 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony3 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/17 transformation m.scFv EPcut+K592009 EPcut colony4 | VF 1.25 VR 1.25 | 9.5 | 2×KAPA HiFi Fast Hotstart /12.5 | 25 |
2/18
PCR (Steps)
Uchino
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 2min | 1 |
Denature | 95℃ | 10sec | 30 |
Annealing | 55℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
2/18
PCR (Purification)
Michimori, Kim
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony1 | 49.8 | 1.92 | 0.08 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony2 | 41.1 | 1.81 | 1.90 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony3 | 47.2 | 1.87 | 1.99 |
2/18 PCR m.scFv EPcut+K592009 EPcut colony4 | 47.5 | 1.87 | 1.93 |
2/19
PCR (Purification)
Yamamoto
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
BAN-cex/(25) | 161.1 | 1.88 | 2.13 |
BAN-clos/(25) | 217.9 | 1.86 | 2.10 |
INP-cex/(25) | 158.6 | 1.86 | 1.98 |
INP-clos/(25) | 163.6 | 1.85 | |
2/19
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
BAN-cex | 12.4 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.6 | |
BAN-clos | 9.2 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 15.8 | |
INP-cex | 12.6 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.4 | |
INP-clos | 12.2 | 1 | 0 | 0 | 1 | 10×H/(3) | 0 | 12.8 | |
2/16
Gel Extraction
Kosaka
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | BAN-cex E, Pcut | 30 |
2 | BAN-clos E, Pcut | 30 |
3 | INP-cex E, Pcut | 30 |
0 | 1kb ladder | 5 |
![](https://static.igem.org/mediawiki/2016/6/65/T--Kyoto--2_19_No18_Gel_Extraction_Kosaka_09_24_12_03_17.1.jpg)
2/19
Gel Extraction
Egashira, Wan, Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 2/19 Restriction Enzyme INP-clos EPcut | 30 |
3 | 2/19 Restriction Enzyme E1010 EPcut | 30 |
4 | blank | - |
![](https://static.igem.org/mediawiki/2016/6/6d/T--Kyoto--2_19_No18_Gel_Extraction_Notsu_10_15_08_27_59.1.jpg)
2/16
PCR (Purification)
Wan
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
2/19 Restriction Enzyme BAN-cex E, Pcut | 9.6 | 1.85 | 0.84 |
〃BAN-clos E, Pcut | 18.9 | 1.83 | 0.71 |
〃INP-cex E, Pcut | 14.9 | 1.77 | 1.46 |
〃INP-clos E, Pcut | 13.4 | 1.73 | 1.67 |
〃E1010 E, Pcut | 19.6 | 1.86 | |
2/19
Transformation
Wan, Egashira, Kim
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
insertDNA/ul | VectoeDNA/ul | ligation high | total |
BAN-cex/4 | E1010cpsb1c3/2 | 6 | 12 |
BAN-clos/3 | 〃/3 | 6 | 12 |
INP-cex/4.5 | 〃/1.5 | 6 | 12 |
INP-clos/4.5 | 〃/1.5 | 6 | |
2/19
Transformation
Wan
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/19 Ligation BAN-cex+E1010 | 10 | DH5a/(20) | LB(CP) |
〃BAN-clos+E1010 | 10 | DH5a/(20) | LB(CP) |
〃INP-cex+E1010 | 10 | DH5a/(20) | LB(CP) |
〃INP-clos+E1010 | 10 | DH5a/(20) | |
2/19
Restriction Enzyme Digestion
Michimori, Kim
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/18 purification m.scFv EPcut+K592009 EPcut colony1 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony2 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony3 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/18 purification m.scFv EPcut+K592009 EPcut colony4 | 12.5 | 0.5 | 0 | 0 | 0.5 | 10×H /1.5 | 0 | 0 | 15 |
2/19
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 2/19 restricion enzyme digestion colony1 /10 | |
3 | 2/19 restricion enzyme digestion colony2 /10 | |
4 | 2/19 restricion enzyme digestion colony3 /10 | |
5 | 2/19 restricion enzyme digestion colony4 /10 | |
2/19
Liquid Culture
Egashira, Wan
Name | medium |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4 | LB(CP) |
2/19
Colony PCR (typeA)
Yamada
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/19 transformation BAN-His-CBDcex colony1 | vf 1.25 vr 1.25 | 10 | 12.5 | 25 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/19 transformation BAN-His-CBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/19 transformation INP-His-CBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃 colony2 | 〃 | 〃 | 〃 | 〃 |
〃 colony3 | 〃 | 〃 | 〃 | 〃 |
2/22
Liquid Culture
Yamada
Name | medium |
2/22 PCR BAN-His-cex colony1 | CP/5ml |
〃colony2 | 〃 |
〃colony3 | 〃 |
2/22 PCR BAN-His-clos colony3 | |
2/22
Miniprep
Egashira, Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony2 | 137.4 | 1.89 | 2.12 |
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony3 | 164.0 | 1.89 | 2.09 |
2/19 liquid culture m.scFv EPcut+K592009 EPcut colony4 | 138.1 | 1.86 | 2.16 |
2/19
Ligation
Wan, Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
insertDNA/ul | VectoeDNA/ul | ligation high | total |
BAN-cex/4 | E1010cpsb1c3/2 | 6 | 12 |
BAN-clos/3 | 〃/3 | 6 | 12 |
INP-cex/4.5 | 〃/1.5 | 6 | 12 |
INP-clos/4.5 | 〃/1.5 | 6 | |
2/23
Transformation
Wan
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/23 Ligation BAN-cex+E1010 | 2 | DH5a/(20) | LB(CP) |
〃clos〃 | 2 | 〃 | 〃 |
〃INP-cex〃 | 2 | 〃 | 〃 |
〃INP-clos〃 | 2 | 〃 | |
2/23
Colony PCR (typeA)
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/23 transformation BAN-His-CBDclos colony1 | vf1.25 vr1.25 | 10 | 12.5 | 25 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
2/23 transformation INP-His-CBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
2/23 transformation INP-His-CBDclos colony1 | 〃 | 〃 | 〃 | |
2/24
Miniprep
Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/22 liqid culture BAN-Hi-cex colony1 | 45.3 | 1.88 | 2.08 |
〃colony2 | 47.1 | 1.89 | 1.98 |
〃colony3 | 43.8 | 1.96 | 2.08 |
2/22 BAN-His-clos colony3 | 53.7 | 1.82 | |
2/24
Liquid Culture
Egashira
Name | medium |
2/23 transformation BAN-clos colony1 | LB(CP/5ml) |
〃colony2 | 〃 |
〃colony3 | 〃 |
2/23 transformation INP-cex colony2 | 〃 |
〃colony3 | 〃 |
2/23 transformation INP-clos colony1 | |
2/25
Miniprep
Egashira, Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/23 transformation BAN-His-CBDclos colony1 | 79.9 | 1.89 | 2.15 |
2/23 transformation BAN-His-CBDclos colony2 | 68.0 | 1.88 | 2.02 |
2/23 transformation BAN-His-CBDclos colony3 | 80.7 | 1.94 | 2.18 |
2/23 transformation INP-His-CBDcex colony2 | 106.0 | 1.91 | 2.17 |
2/23 transformation INP-His-CBDcex colony3 | 136.7 | 1.93 | 2.23 |
2/23 transformation INP-His-CBDclos colony1 | 183.7 | 1.91 | 2.21 |
2/25
Sequence
Michimori
Sample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
Bcx 1-F | 2/24 miniprep BAN-His-CBDcex colony1 /6.6 | VF /0.2 | 3.2 | 10 |
Bcx 1-R | 2/24 miniprep BAN-His-CBDcex colony1 /6.6 | VR /0.2 | 3.2 | 10 |
Bcx 2-F | 2/24 miniprep BAN-His-CBDcex colony2 /6.4 | VF /0.2 | 3.4 | 10 |
Bcx 2-R | 2/24 miniprep BAN-His-CBDcex colony2 /6.4 | VR /0.2 | 3.4 | 10 |
Bcl1-F | 2/25 miniprep BAN-His-CBDclos colony1 /3.8 | VF /0.2 | 6.0 | 10 |
Bcl1-R | 2/25 miniprep BAN-His-CBDclos colony1 /3.8 | VR /0.2 | 6.0 | 10 |
Bcl2-F | 2/25 miniprep BAN-His-CBDclos colony3 /3.7 | VF /0.2 | 6.1 | 10 |
Bcl2-R | 2/25 miniprep BAN-His-CBDclos colony3 /3.7 | VR /0.2 | 6.1 | 10 |
Icx1-F | 2/25 miniprep INP-His-CBDcex colony2 /2.8 | VF /0.2 | 7.0 | 10 |
Icx1-R | 2/25 miniprep INP-His-CBDcex colony2 /2.8 | VR /0.2 | 7.0 | 10 |
Icx2-F | 2/25 miniprep INP-His-CBDcex colony3 /2.2 | VF /0.2 | 7.6 | 10 |
Icx2-R | 2/25 miniprep INP-His-CBDcex colony3 /2.2 | VR /0.2 | 7.6 | 10 |
Icl1-F | 2/25 miniprep INP-His-CBDclos colony1 /1.6 | VF /0.2 | 8.2 | 10 |
Icl1-R | 2/25 miniprep INP-His-CBDclos colony1 /1.6 | VR /0.2 | 8.2 | 10 |
2/25
Liquid Culture
Yamada
Name | medium |
2/19 transformation BAN-His-cex colony1 | LB(CP/5ml) |
2/19 transformation BAN-His-cex colony2 | LB(CP/5ml) |
2/23 transformation BAN-His-clos colony1 | LB(CP/5ml) |
2/23 transformation BAN-His-clos colony3 | LB(CP/5ml) |
2/23 transformaation INP-His-cex colony2 | LB(CP/5ml) |
2/23 transformation INP-His-cex colony3 | LB(CP/5ml) |
INP-His-clos colony1 | |
2/25
Sequence
Michimori
Sample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
S2-F | 2/22 miniprep scFv colony2 /2.2 | VF /0.2 | 7.6 | 10 |
S2-R | 2/22 miniprep scFv colony2 /2.2 | VR /0.2 | 7.6 | 10 |
S3-F | 2/22 miniprep scFv colony3 /1.8 | VF /0.2 | 8.0 | 10 |
S3-R | 2/22 miniprep scFv colony3 /1.8 | VR /0.2 | 8.0 | 10 |
S4-F | 2/22 miniprep scFv colony4 /2.2 | VF /0.2 | 7.6 | 10 |
S4-R | 2/22 miniprep scFv colony4 /2.2 | VR /0.2 | 7.6 | 10 |
114C-R
2/25
Liquid Culture
Egashira, Kim, Yamada
Name | medium |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony2 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony3 | LB(CP) |
2/17 transformaion m.scFv EPcut+K592009 EPcut colony4 | LB(CP) |
2/26
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
J23100 | VF/ 0.75 | J23100+RBSRv | | 11 | PrimeSTARMax Premix/ 12.5 | 25 |
J23108 | VF/ 0.75 | J23108+RBSRv | | 11 | PrimeSTARMax Premix/ 12.5 | 25 |
J23114 | VF/ 0.75 | J23114+RBSRv | | 11 | | 25 |
2/26
PCR (Target)
Li
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
J23100 iGEM Part | VF /0.75 | J23100+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
J23108 iGEM Part | VF /0.75 | J23108+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
J23114 iGEM Part | VF /0.75 | J23114+RBS Rv /0.75 | - | 11 | PrimeSTAR Max Premix /12.5 | 25 |
2/26
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | - |
2 | 2/26 PCR J23100 /5 | 193+α |
3 | 2/26 PCR J23108 /5 | 193+α |
4 | 2/26 PCR J23114 /5 | 193+α |
2/26
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
J23100 iGEM Part /0.1 | VF 0.75 | J23100+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
J23108 iGEM Part /0.1 | VF 0.75 | J23108+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
J23114 iGEM Part /0.1 | VF 0.75 | J23114+RBS Rv /0.75 | - | 10.9 | KAPA HiFi /12.5 | 25 |
2/26
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 2/26 PCR J23100 | 25 |
3 | 2/26 PCR J23108 | 25 |
4 | 2/26 PCR J23114 | |
2/29
Gel Extraction(Measurement)
Egashira, Wan
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
2/26 PCR J23100 | 1.5 | 2.71 | 0.09 |
2/26 PCR J23108 | 3.6 | 18.64 | 0.01 |
2/26 PCR J23114 | 1.7 | 3.05 | 0.13 |
2/29
PCR (Target)
Wan, Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
J23100 iGEM Part /0.2 | VF /1.5 | J23100+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
J23108 iGEM Part /0.2 | VF /1.5 | J23108+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
J23114 iGEM Part /0.2 | VF /1.5 | J23114+RBS Rv /1.5 | - | 21.8 | KAPA HiFi /25 | 50 |
3/1
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | - |
2 | 3/1 PCR J23100 /5 | about 200 |
3 | 3/1 PCR J23108 /5 | about 200 |
4 | 3/1 PCR J23114 /5 | about 200 |
5 | 3/1 PCR m.scFv colony3 /5 | 949 |
3/1
Electrophoresis
Kim, Yamada
Lane | sample/(µl) | Length(bp) |
1 | 100bp ladder /5 | - |
2 | 3/1 OE-PCR J23100 + m.scFv colony3 /2 | about 1200 |
3 | 3/1 OE-PCR J23108 + m.scFv colony3 /2 | about 1200 |
4 | 3/1 OE-PCR J23114 + m.scFv colony3 /2 | about 1200 |
3/1
PCR (Purification)
Kim, Yamada
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/1 OE-PCR J23100 + m.scFv colony3 | 180.5 | 1.89 | 1.83 |
3/1 OE-PCR J23108 + m.scFv colony3 | 183.3 | 1.87 | 1.96 |
3/1 OE-PCR J23114 + m.scFv colony3 | 178.8 | 1.87 | 2.08 |
3/1
PCR (Target)
Kim, Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/1 PCR J23100 /1 + 3/1 PCR m.scFv colony3 /0.5 | VF /0.5 | VR /0.75 | - | 9.5 | Prime Star Premix /12.5 | 25 |
3/1 PCR J23108 /1 + 3/1 PCR m.scFv colony3 /0.5 | VF /0.5 | VR /0.75 | - | 9.5 | Prime Star Premix /12.5 | 25 |
3/1 PCR J23114 /2 + 3/1 PCR J23114 /0.5 | VF /0.5 | VR /0.75 | - | 8.5 | Prime Star Premix /12.5 | 25 |
3/1
Gel Extraction
Kim, Yamada
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | |
2 | 3/1 EPcut J23114 + m.scFv | 20 |
3 | 3/1 EPcut J23108 + m.scFv | 20 |
4 | 3/1 EPcut J23100 + m.scFv | 20 |
3/1
Restriction Enzyme Digestion
Li, Kim, Yamada
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/1 OE-PCR J23100 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/1 OE-PCR J23108 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/1 OE-PCR J23114 + m.scFv colony3 | 11 | 1 | 0 | 0 | 1 | 10×H /3 | - | 14 | 30 |
3/2
PartsAwaking
Michimori, Kim, Yamada
Description | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
f1 origin | K314110 | pSB1C3 | 1A | 2016 | 1 | 10 |
T1 terminator from E. coli rrnB | K731722 | pSB1C3 | 1B | 2016 | 1 | 10 |
Promoter of the CaiF protein | K398326 | pSB1C3 | 1C | 2016 | 1 | 10 |
3/2
Transformation
Michimori
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
3/2 Parts Awakening K314110 | 10 | DH5α /20 | LB(CP) |
3/2 Parts Awakening K731722 | 10 | DH5α /20 | LB(CP) |
3/2 Parts Awakening K398326 | 10 | DH5α /20 | LB(CP) |
3/2
Gel Extraction(Measurement)
Yamada
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
J23100+m.scFv 3/1 EPcut | 17.7 | 1.90 | 1.68 |
J23108+m.scFv 3/1 EPcut | 11.9 | 2.04 | 0.26 |
J23114+m.scFv 3/1 EPcut | 16.8 | 1.97 | 1.79 |
3/2
Restriction Enzyme Digestion
Wan
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
E0020 | 40 | 2 | 0 | 0 | 2 | 10×H /6 | - | 10 | 60 |
3/2
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
J23100+pSB1C3 | 2 | DH5α /20 | LB(CP) |
J23108+pSB1C3 | 2 | DH5α /20 | LB(CP) |
J23114+pSB1C3 | 2 | DH5α /20 | LB(CP) |
3/3
PCR (Target)
Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/9 miniprep E1010 /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/24 miniprep BAN-His-CBDcex colony2 /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep BAN-His-CBDclos /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep INP-His-CBDcex /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
2/25 miniprep INP-His-CBDclos /1 | VF 0.75 | VR 0.75 | - | 10 | Prime Star premix 12.5 | 25 |
3/3
Gel Extraction(Measurement)
Egashira
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
3/2 EPcut E0020 | 23.1 | 1.85 | 1.69 |
3/3
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | |
2 | 3/2 EPcut E0020 (pSB1C3) | 20 |
3/3
Ligation
Wan
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23100+m.scFv EPcut /12 | 16 | 32 |
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23108+m.scFv EPcut /12 | 20 | 40 |
3/3 Gel Extraction E0020 EPcut /4 | 3/2 Gel Extraction J23114+m.scFv EPcut /12 | 16 | 32 |
3/3
Transformation
Wan, Kim
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
J23100+PSB1C3 | 2 | DH5alpha/ 20 | LB(CP) |
J23108+PSB1C3 | 2 | DH5alpha/ 20 | LB(CP) |
J23114+PSB1C3 | 2 | DH5alpha | LB(CP) |
3/3
Liquid Culture
Wan, Kim
Name | medium |
K314110 colony1 | LB(CP) |
K731722 colony1 | LB(CP) |
K398326 colony1 | LB(CP) |
3/4
Transformation
Li, Notsu
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
J23100+pSB1C3 | 10 | DH5α /20 | LB(CP) |
J23108+pSB1C3 | 10 | DH5α /20 | LB(CP) |
J23114+pSB1C3 | 10 | DH5α /20 | LB(CP) |
3/7
Miniprep
Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
2/25 liquid culture BAN-His-CBDcex colony2 | 71.4 | 1.83 | 1.43 |
2/25 liquid culture BAN-His-CBDclos colony1 | 61.6 | 1.83 | 1.90 |
2/25 liquid culture INP-His-CBDcex colony3 | 83.4 | 1.94 | 1.92 |
2/25 liquid culture INP-His-CBDclos colony1 | 100.7 | 1.87 | 1.44 |
3/7
Transformation
Wan
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
3/7 miniprep BAN-His-CBDcex | 2 | DH5α /20 | LB(CP) |
3/7 miniprep BAN-His-CBDclos | 2 | DH5α /20 | LB(CP) |
3/7 miniprep INP-His-CBDcex | 2 | DH5α /20 | LB(CP) |
3/7 miniprep INP-His-CBDclos | 2 | DH5α /20 | LB(CP) |
| | | |
3/7
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
3/3 ligation J23100+pSB1C3 | | DH5α /20 | LB(CP) |
3/3 ligation J23108+pSB1C3 | | DH5α /20 | LB(CP) |
3/3 ligation J23114+pSB1C3 | | DH5α /20 | LB(CP) |
3/7
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
3/4 transformtion J23100 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23100 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23108 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23108 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23114 colony1 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/4 transformtion J23114 colony2 | VF 1.25 VR 1.25 | 10 | KAPA2G /12.5 | 25 |
3/7
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 2min | 1 |
Denature | 95℃ | 15sec | 35 |
Annealing | 55℃ | 15sec | 35 |
Extension | 72℃ | 5sec | 35 |
Final Elongation | 72℃ | 10min | 1 |
Final Holding | 4℃ | - | - |
3/7
Electrophoresis
Wan, Yamamoto
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 3/7 colony PCR J23100 colony1 /10 | 1000+α |
3 | 3/7 colony PCR J23100 colony2 /10 | 1000+α |
4 | 3/7 colony PCR J23108 colony1 /10 | 1000+α |
5 | 3/7 colony PCR J23108 colony2 /10 | 1000+α |
6 | 3/7 colony PCR J23114 colony1 /10 | 1000+α |
7 | 3/7 colony PCR J23114 colony2 /10 | 1000+α |
3/7
PCR (Steps)
Yamada
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Extension | 72℃ | 5min | 35 |
Final Elongation | 4℃ | - | - |
Final Holding | 4℃ | - | - |
3/7
PCR (Target)
Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/1 purification J23100 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23100 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23108 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23108 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23114 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/1 purification J23114 | VF /3 | VR /3 | - | 44 | KAPA HiFi /50 | 100 |
3/7
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
3/7 transformation BAN-CBDcex colony1 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-cex colony2 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-clos colony1 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation BAN-closcolony1 | VF 1.5 VR1.25 | 10 | 12.5 | |
3/7 transformation INP-cex colony2 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-cex colony2 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-clos colony1 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/7 transformation INP-clos colony 2 | VF 1.5 VR1.25 | 10 | 12.5 | 25 |
3/8
PCR (Purification)
Uchino
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/7 PCR J23100 | 423.3 | 1.84 | 2.20 |
3/7 PCR J23108 | 439.1 | 1.85 | 2.11 |
3/7 PCR J23114 | 451.2 | 1.81 | 1.91 |
3/8
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/8 purification J23100 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23108 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23114 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8
Gel Extraction(Measurement)
Li, Kim, Yamada
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
J23114 3/8 EPcut | 13.7 | 3.62 | 0.03 |
J23108 3/8 EPcut | 15.4 | 1.83 | 1.14 |
J23100 3/8 EPcut | 14.5 | 1.79 | 1.03 |
3/9
Miniprep
Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
K731722 | 67.2 | 1.70 | 1.02 |
K314110 | 28.9 | 1.86 | 1.14 |
K398326 | 95.6 | 1.64 | 0.94 |
3/9
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/1 miniprep K314110 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/1 miniprep K731722 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/1 miniprep K398326 | 25 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 0 | 30 |
3/9
Restriction Enzyme Digestion
Li
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/8 purification J23100 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23108 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23114 | 5 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20 | 30 |
3/8 purification J23100 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
3/8 purification J23108 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
3/8 purification J23114 | 20 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 5 | 30 |
| | | | | | | | | 30 |
3/11
Gel Extraction
Yamada
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 3/10 E.P. cut J23100+scFv | 15 |
3 | 3/10 E.P. cut J23100+scFv | 15 |
4 | 3/10 E.P.cut J23108+scFv | 15 |
5 | 3/10 E.P.cut J23108+scFv | 15 |
6 | 3/10 E.P.cut J23114+scFv | 15 |
7 | 3/10 E.P.cut J23114+scFv | 15 |
8 | 3/9 E.P. cut J23100+scFv | 15 |
9 | 3/9 E.P. cut J23100+scFv | 15 |
10 | 3/9 E.P. cut J23119 scFv | 15 |
11 | 3/9 E.P. cut J23119 scFv | 15 |
12 | 3/9 E.P.cut J23114+scFv | |
3/11
Gel Extraction
Yamada
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kbp ladder | 15 |
2 | 3/9 E.P. cut K314110 | 15 |
3 | 3/9 E.P. cut K314110 | 15 |
4 | 3/9 E.P cut K731722 | 15 |
5 | 3/9 E.P cut K731722 | 15 |
6 | 3/9 E.P cut K7398326 | 15 |
7 | 3/9 E.P cut K7398326 | 15 |
8 | 3/9 E.P> cut J23114+scFv | |
3/11
PCR (Purification)
Yoshimoto
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/11 Gel Ex J23108+scFv | 18.9 | 1.90 | 0.19 |
3/11 Gel Ex J23108+scFv | 7.0 | 1.92 | 0.57 |
3/11 Gel Ex J23114+scFv | 5.3 | 2.37 | 0.25 |
3/11 Gel Ex J23100+scFv | 39.2 | 1.93 | 1.29 |
3/11 Gel Ex J23108+scFv | 33.6 | 1.82 | 0.68 |
3/11 Gel Ex J23114+scFv | 27.8 | 1.86 | 1.25 |
3/11 Gel Ex pSCIC3 | 29.0 | 1.86 | |
3/11
Ligation
Kim
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23100+scfv/ 5.6 | 10.6 | 21.2 |
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23108+scfv/ 5 | 11.5 | 23 |
3/11 Gel Extraction PSB1C3/ 5 | 3/11 Gel Extraction J23114+scfv/ 5 | 12.8 | |
3/11
Transformation
Kim
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
3/11 Ligation PSB1C3+J23100+scFV | | DH5α /20 | LB |
3/11 Ligation PSB1C3+J23108+scFv | | DH5α /20 | LB |
3/11 Ligation PSB1C3+J23114+scFv | | DH5α /20 | LB |
3/7
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder | |
2 | 3/8 PCR BANcex colony1 | 515 |
3 | 3/8 PCR BAN-cex colony2 | 515 |
4 | 3/8 PCR BAN-clos colony1 | 487 |
5 | 3/8 PCR BAN-clos-colony2 | 487 |
6 | 3/8 PCR INP-cex colony 1 | 1405 |
7 | 3/8 PCR INP-cex colony 2 | 1405 |
8 | 3/8 PCR INP-clos colony 1 | 1378 |
9 | 3/8 PCR INP-clos colony 2 | 1378 |
3/14
PCR (Target)
Uchino, Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/25 INPcex colony2 | VF 0.75 CBDcex ctl. Rv0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/25 INPcex colony2 | CBDcex ctl Fw 0.75 VR0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/18 INPclos Gel Extraction | VF 0.75 CBD clos ctl. Rv0.75 | 11 | KAPA HiFi 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl Fw 0.75 VR0.75 | 11 | KAPA HiFi 12.5 | 25 |
3/14
Gel Extraction
Uchino, Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
4 | 3/14PCR INP cex (VF CBD cex ctl.Rv)25 | 5 |
5 | 3/14PCR INPcex (CBD cex ctl.Fw VR) 25 | 5 |
6 | 3/14PCR INP clos(VF CBDclos ctl.Rv)25 | 5 |
7 | 3/14PCR INPclos (CBDclos ctl.Fw VR) 25 | 5 |
![](https://static.igem.org/mediawiki/2016/9/98/T--Kyoto--3_14_No45_Gel_Extraction_Yoshimoto_09_02_09_48_51.1.jpg)
3/15
Restriction Enzyme Digestion
Notsu
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/7 miniprep BAN-His-CBDcex colony2 | 30 | | | 1 | 1 | 4 | | 4 | 40 |
3/7miniprep BAN-His-CBDclos colony1 | 30 | | | 1 | 1 | 4 | | 4 | 40 |
3/7miniprep INP-His-CBDcex colony3 | 30 | | | 1 | 1 | 4 | | 4 | 40 |
3/'miniprep INP-His-CBDcloscolony 1 | 40 | | | 1 | 1 | 5 | | 3 | 50 |
2/9 miniprep J23105 | 61 | | 1 | | | | | 0 | 70 |
3/14
OE-PCR (typeA)
Kim
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
3/14 Gel Ex INP cex 0.5 VR ct Fw + 3/14 Gel Ex INP cex 0.5 VF ct Rv | VF1.5 VR1.5 | 21 | 25 | 50 |
3/14 Gel Ex INP clos 0.5 VR ct Fw + 3/14 Gel Ex INP clos 0.5 VF ct Rv | VF1.5 VR1.5 | 21 | 25 | 50 |
3/14
PCR (Steps)
Kim
Steps | Temparature | Time | Cycle |
Pre Denature | 95℃ | 5min | 1 |
Denature | 78℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Extension | 72℃ | 45sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
3/14
Colony PCR (typeA)
Yoshimoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/8 INP His9 | VF 0.75 scFv ctl. Rv 0.75 | 11 | 12.5 | 25 |
2/8 INP His9 | scFv ctl Fw 0.75 VR 0.75 | 11 | 12.5 | 25 |
2/25 INPcex colony2 | VF 0.75 CBDcex ctl. Rv0.75 | 11 | 12.5 | 25 |
2/25 INPcex colony2 | CBDcex ctl. Fw 0.75 VR 0.75 | 11 | 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl. Rv 0.75 VF 0.75 | 11 | 12.5 | 25 |
2/18 INPclos Gel Extraction | CBDclos ctl Fw 0.75 | 11 | 12.5 | |
3/14
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 3/14 PCR INP His9 (VF scFv.ctl. Rv) | 25 |
3 | 3/14 PCR INP His9 (scFv ctl.Fw VR) | |
![](https://static.igem.org/mediawiki/2016/1/11/T--Kyoto--3_14_No173_Gel_Extraction_Yoshimoto_09_16_10_54_56%2C1.jpg)
3/15
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder 5 | |
2 | 3/14 OE PCR CBDcex ctl 50 | 10 |
3 | 3/14 OEPCR CBDclos ctl 50 | |
3/15
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/25 INPcex colony2 | VF /1.5 | CBDcex.ctl.RV /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPcex colony2 | CBDcex.ctl.Fw /1.5 | VR /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPclos colony1 | VF /1.5 | CBDclos.ctl.Rv /1.5 | - | 22 | KAPA HiFi /25 | 50 |
2/25 INPclos colony1 | CBDclos.ctl.Fw /1.5 | VR /1.5 | - | 22 | KAPA HiFi /25 | 50 |
3/15
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
4 | 3/15 PCR INPcex(VF 20, CBDcex ctl.Rv 20) | 4 |
5 | 3/15 PCR INPcex(CBDcex ctl.Fw20, VR 20) | 4 |
6 | 3/15 PCR INPclos(VF 20 CBDclos ctl.Rv 20) | 4 |
7 | 3/15 PCR INPclos(CBDclos ctl.Fw20, VR 20) | 4 |
3/15
Liquid Culture
Li, Uchino
Name | medium |
3/11 scFv J23100 colony1,4 colony2,3(plate2) | LB(CP) 5×2 |
3/11 scFv J23108 colony 2(plate1) colony(plate2) | LB(CP) 5×2 |
3/14 scFv J23114 colony 1,2,4(plate1) colony1,2,(plate2) | |
3/16
PCR (Purification)
Kim, Yamada
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/15 Gelex CBDcex ctl. Fw VR | 55.3 | 1.95 | 0.11 |
3/15 Gelex CBDcex VF ctl.RV | 8.2 | 1.74 | 1.04 |
3/15Gelex CBDclos ctl Fw VR | 41.2 | 1.85 | 1.51 |
3/15Gelex CBDclos VF ctl RV | 10.6 | 1.56 | 0.60 |
3/15Gelex BANcexSP | 34.9 | 1.70 | 1.84 |
3/15Gelex BANclosSP | 32.0 | 1.95 | 0.07 |
3/15 Gelex INPcex SP | 40.3 | 1.76 | 1.76 |
3/15Gelex INPclosSP | 106.8 | 1.83 | 0.19 |
3/16
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
3 | 3/15 PCR INPcex(VF 20, CBDcex ctl, RV 20) | 4 |
4 | 3/15 PCR INPclos(VF 20, CBDclos ctl, RV 20) | |
3/16
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
4 | CBDcex ctl | |
5 | CBDclos ctl | |
3/16
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/15 Gel Ex INPcex 0.5(VF ctl.RV) 3/15 Gel Ex INPcex0.2(ctl.Fw VR) | VF1.25 | VR1.25 | KAPA taq 12.5 | 9.3 | | 25 |
3/15 GEl EX INPclos 0.5(VF ctl RV) 3/15Gel Ex INPclos0.2(ctl.Fw VR) | VF1.25 | | KAPA taq 12.5 | 9.3 | | 25 |
3/16
PCR (Target)
Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/14 Gel Extraction INP-His9 VR/ct/Rv /0.5 + 3/14 Gel Extraction INP-His9 /0.5 | VF /1.5 | VR /1.5 | - | 21 | 2×KAPA HiFi /25 | 50 |
3/16
PCR
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | KAPA HiFi/(µl) | Total/(µl) |
2/8 INP HIs9 | VF 1.5 scFvctl. RV1.5 | 22 | 25 | 50 |
2/8 INP His9 | VR1.5 scFvctl. Fw 1.5 VR1.5 | 22 | 25 | |
3/16
Miniprep
Egashira, Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
J23100 scFv c1-p1 | 135.7 | 1.85 | 1.76 |
J23100 scFv C4-p1 | 135.1 | 1.86 | 1.85 |
J23100 scFv c3-p2 | 155.0 | 1.87 | 2.04 |
J23100 scFv c3-p2 | 205.9 | 1.76 | 1.32 |
J23108 scFv c2-p1 | 166.5 | 1.88 | 2.06 |
J23108 scFvc1-p2 | 95.7 | 1.84 | 1.83 |
J23114 scFv c1-p1 | 145.7 | 1.80 | 1.99 |
J23114 scFv c2-p1 | 111.7 | 1.90 | 2.16 |
J23114 scFv c4-p1 | 169.2 | 1.90 | 2.11 |
J23114 scFv c1-p2 | 127.4 | 1.87 | 2.00 |
J23114 scFv c2-p2 | 136.2 | 1.90 | 2.13 |
3/16
Sequence
Michimori
Sample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
100A-F | 3/15 miniprep J23100 scFv c1-p1 /2.2 | VF /0.2 | 7.6 | 10 |
100A-R | 3/15 miniprep J23100 scFv c1-p1 /2.2 | VR /0.2 | 7.6 | 10 |
100B-F | 3/15 miniprep J23100 scFv c4-p1 /2.2 | VF /0.2 | 7.6 | 10 |
100b-R | 3/15 miniprep J23100 scFv c4-p1 /2.2 | VR /0.2 | 7.6 | 10 |
100C-F | 3/15 miniprep J23100 scFv c2-p2 /1.9 | VF /0.2 | 7.9 | 10 |
100C-R | 3/15 miniprep J23100 scFv c2-p2 /1.9 | VR /0.2 | 7.9 | 10 |
100D-F | 3/15 miniprep J23100 scFv c3-p2 /1.5 | VF /0.2 | 8.3 | 10 |
100D-R | 3/15 miniprep J23100 scFv c3-p2 /1.5 | VR /0.2 | 8.3 | 10 |
108A-F | 3/15 miniprep J23108 scFv c2-p1 /1.8 | VF /0.2 | 8.0 | 10 |
108A-R | 3/15 miniprep J23108 scFv c2-p1 /1.8 | VR /0.2 | 8.0 | 10 |
108B-F | 3/15 miniprep J23108 scFv c1-p2 /3.1 | VF /0.2 | 6.7 | 10 |
108B-R | 3/15 miniprep J23108 scFv c1-p2 /3.1 | VR /0.2 | 6.7 | 10 |
114A-F | 3/15 miniprep J23114 scFv c1-p1 /2.1 | VF /0.2 | 7.7 | 10 |
114A-R | 3/15 miniprep J23114 scFv c1-p1 /2.1 | VR /0.2 | 7.7 | 10 |
114B-F | 3/15 miniprep J23114 scFv c2-p1 /2.7 | VF /0.2 | 7.1 | 10 |
114B-R | 3/15 miniprep J23114 scFv c2-p1 /2.7 | VR /0.2 | 7.1 | 10 |
114C-R
3/16
Sequence
Michimori
Sample | DNA template/(µl) | Primer/(µl) | MilliQ(µl) | Total(µl) |
114C-F | 3/15 miniprep J23114 scFv c4-p1 /1.8 | VF /0.2 | 8.0 | 10 |
114C-R | 3/15 miniprep J23114 scFv c4-p1 /1.8 | VR /0.2 | 8.0 | 10 |
114C-F | 3/15 miniprep J23114 scFv c1-p2 /2.4 | VF /0.2 | 7.4 | 10 |
114C-R | 3/15 miniprep J23114 scFv c1-p2 /2.4 | VR /0.2 | 7.4 | 10 |
114C-F | 3/15 miniprep J23114 scFv c2-p2 /2.2 | VF /0.2 | 7.6 | 10 |
114C-R | 3/15 miniprep J23114 scFv c2-p2 /2.2 | VR /0.2 | 7.6 | 10 |
114C-R
3/16
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
2 | 3/15 PCR INP His9(VF scFv CH. Rv) | 20 |
3 | 3/15 PCR INP His9 (scFv ctl, Fw VR) | |
3/16
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 3/15 PCR INP His9 (VF scFv ctl RV) | |
3/16
Gel Extraction
Egashira
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | |
2 | scFv (VF ctl.Rv) | |
3 | | |
3/16
OEPCR (Target)
Egashira
Sample1/(µl) | Sample2/(µl) | Primer/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/15 Gel Extraction scfv (VF ctl.Rv)/ 0.5 | 3/15 Gel Extraction scfv (ctl.Fw VR) | VF/ 1.25, VR/1.25 | | 9.3 | KAPA Taq/ 12.5 | 25 |
3/16
PCR
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
scFv (VF ctl.RV) 0.5 | VF 1.25 ctl.Rv) 1.25 | 9.5 | 12.5 | 25 |
3/16
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.25+3/16 Gel Extraction scFv(ctl.Fw.VR) /0.25 | VF /1.25 | VR /1.25 | - | 9.5 | 2×KAPA HiFi /12.5 | 25 |
3/17
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/16 Gel Ex scFv(VF ctl.Rv)0.25, 3/15 Gel EX CBDcex(ctl.Fw VR) 0.25 | VF 1.25 | VR1.25 | KAPA Taq 12.5 | 9.5 | | 25 |
3/16 Gel Ex scFv(VF ctl.Rv) 0.25, Gel Ex CBDclos(ctl,Fw VR) 0.25 | VF 1.25 | VR1.25 | KAPA Taq 12.5 | | | 25 |
3/17
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
3 | 3/16 OEPCR CBDcex ctl 25 | 500 |
4 | 3/16 OEPCR CBDclos ctl 25 | 500 |
3/17
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
2 | CBDcex ctl 25 | |
3 | CBDclos ctl 25 | |
4 | 1kb ladder 5 | |
3/17
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/16 Gel Ex scFv(VF ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 | VF 1.25 | VR1.25 | KAPA 12.5 | 9.8 | | 25 |
3/16 Gel Ex scFv(VF ctl,Rv) 0.1, 3/15 GelEx CBDclos(ctl,Fw VR) 0.1 ctl,Rv)0.1 3/15 Gel Ex CBDcex(ctl,Fw VR) 0.1 | VF 1.25 | VR1.25 | KAPA 12.5 | 9.8 | | 25 |
3/17
Miniprep
Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
3/8 Liquid Culture BAN-His-cex | 39.3 | 1.91 | 1.67 |
3/8 Liquid Culture BAN-His=clos | 45.1 | 1.84 | 1.44 |
3/17
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Pre Denature | 95℃ | 3min | 1 |
Denature | 95℃ | 15sec | 40 |
Annealing | 55℃ | 15sec | 40 |
Elongation | 72℃ | 1min | 40 |
Final Elongation | 72℃ | 1min | 1 |
Final Holding | 4℃ | - | - |
3/17
PCR (Target)
Yamamoto
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/17 miniprep BAN-His-cex | CBDcexFw 1.25 | VR1.25 | 12.5 | 9 | | 25 |
3/17 miniprep BAN-His-clos | CBDclosFw 1.25 | VR1.25 | 12.5 | 9 | | 25 |
2/9 miniprep J23105_1 | VF 1.25 | RFP-cex RV 1.25 | 12.5 | 9 | | 25 |
2/9 miniprep J23105_1 | VF 1.25 | RFP-clos Rv 1.25 | 12.5 | 9 | | 25 |
3/17
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder 5 | |
2 | | |
3/17
PCR (Target)
Egashira
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/16 Gel Extraction scFv(VF.ctl.Rv) /0.1+3/15 Gel Extraction scFv(ctl.Fw.VR) /0.1 | VF /1.25 | VR /1.25 | - | 9.8 | 2×KAPA HiFi /12.5 | 25 |
3/18
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 3/10 PCR BAN-cex CBDcexFW VR | 25 |
3 | 3/10 PCR BAN-clos CBDclosFw VR | 25 |
4 | 3/10 PCR J23105 VF RFPcexRv | 25 |
5 | 3/10 PCRJ23105 VF RFPclos | 25 |
![](https://static.igem.org/mediawiki/2016/6/62/T--Kyoto--3_18_No63_Gel_Extraction_Yoshimoto_09_03_12_29_48.1.jpg)
3/18
Gel Extraction(Measurement)
Yamamoto
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
3/17 PCR BAN-CBDcex CBDcex.Fw+Rv | 127.7 | 1.90 | 1.57 |
3/17 PCR BAN-CBDclos CBDclos.Fw+VR | 93.4 | 1.92 | 2.19 |
3/17 PCR J23105 VF+RFP-CBDcex.Rv | 104.0 | 1.84 | 1.50 |
3/17 PCR J23105 VF+RFP-CBDclos.Rv | 123.0 | 1.87 | 1.84 |
3/18
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Pre Denature | 95℃ | 3min | 1 |
Denature | 95℃ | 15sec | 40 |
Annealing | 60℃ | 15sec | 40 |
Elongation | 72℃ | 1min | 40 |
Final Elongation | 72℃ | 1min | 1 |
Final Holding | 4℃ | - | - |
3/18
PCR (Target)
Yamamoto
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/18 Gel Extraction BAN-His-cex 0.1 cexFw+VR , 3/18Gel Extraction J23105 VF+RFP-cexRv | VF 1.25 | VR 1.25 | KAPATaq 12.5 | 9.8 | | 25 |
3/18 Gel Extraction BAN-His-clos 0.1 closFw+VR , 3/18Gel Extraction J23105 VF+RFP-closRv | VF 1.25 | VR 1.25 | KAPATaq 12.5 | 9.8 | | 25 |
3/18
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | |
2 | 3/18 OEPCR RFP-cex | |
3 | 3/18 OEPCR RFP-clos | |
3/18
PCR (Target)
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/25 INP cex colony2 0.1×2 | VF 0.75×2 ctl RV 0.75 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | ctl Fw 0.75×2 VR 0.75×2 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | VF 0.75×2 VR 0.75×2 | 10.9×2 | 12.5×2 | 25×2 |
2/25 INP cex colony2 0.1×2 | nothing | 12.4 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | VF ctl.Rv 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | ctl.Rv 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | VF 0.75×2 VR 0.75×2 | 10.9 | 12.5×2 | 25×2 |
2/25 INP clos colony1 0.1 | nothing | 12.4 | 12.5×2 | 25×2 |
3/18
Colony PCR (typeA)
Notsu
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INP-His=CNDcex+RFP colony 1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDcex+RFP colony (3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDclos+RFP colony1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDclos+RFP colony 2(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
INP-His-CBDcex+RFP colony1( 3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDcex+RFP colony2 (3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDclos+RFP colony1(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
BAN-His-CBDclos+RFP colony2(3/16 ligation product) | VF 1.25, VR .125 | 10 | 12.5 | 25 |
3/18
Electrophoresis
Kim, Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder 5 | |
2 | 1 kb ladder 5 | |
3 | 2/25 INPcex colony2 10 | |
4 | 2/25 INPcex colony2 10 | |
5 | 2/25 INPcex colony2 10 | |
6 | 2/25 INPcex colony210 | |
7 | 2/25 INPclos colony1 10 | |
8 | 2/25 INPclos colony1 10 | |
9 | 2/25 INPclos colony1 10 | |
10 | 2/25 INPclos colony1 10 | |
11 | 100bp ladder 5 | |
12 | 1kbp ladder 5 | |
3/18
PCR
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INP HIs9 0.1x2 | VF 0.75x2 ctl. Rv 0.75x2 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | ctl.Fw 1.75 VR 0.75 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | VF 0.75 VR 0.75 | 10.9x2 | 12.5x2 | 25x2 |
INP HIs9 0.1x2 | nothing | 12.4 | 12.5x2 | |
3/22
Electrophoresis
Notsu
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /7 | |
2 | INP-His-CBDcex+RFP colony1 (3/19 PCR) /20 | |
3 | INP-His-CBDcex+RFP colony2 (3/19 PCR) /20 | |
4 | blank | |
5 | INP-His-CBDclos+RFP colony1 (3/19 PCR) /20 | |
6 | INP-His-CBDclos+RFP colony2 (3/19 PCR) /20 | |
7 | blank | |
8 | BAN-His-CBDcex+RFP colony1 (3/19 PCR) /20 | |
9 | BAN-His-CBDcex+RFP colony2 (3/19 PCR) /20 | |
10 | blank | |
11 | BAN-His-CBDclos+RFP colony1 (3/19 PCR) /20 | |
12 | BAN-His-CBDclos+RFP colony2 (3/19 PCR) /20 | |
13 | blank | |
14 | 100bp ladder /7 | |
15 | - | |
16 | - | |
17 | - | |
3/22
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
7 | 2/25 INPcexcolony2 (5) 10 | |
8 | 2/25 INPcexcolony2 (6) 10 | |
9 | 2/25 INPcexcolony2 (7) 10 | |
10 | 2/25 INPcexcolony2 (8) 10 | |
11 | 2/25 INPclos colony1(9) 10 | |
12 | 2/25 INPclos colony1(10) 10 | |
13 | 2/25 INPclos colony1(11) 10 | |
14 | 2/25 INPclos colony1(12) 10 | |
16 | 100b ladder 5 | |
17 | 1kb ladder 5 | |
3/22
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
5 | 3/22 PCR CBDcex(VF/ctl.Rv) 10 | |
6 | 3/22 PCR CBDcex(ctl.Fw/VR) 10 | |
7 | 3/22 PCR CBDclos(VP/ctl.RV) 10 | |
8 | 3/22 PCR CBDclos(ctl.Fw/VR) 10 | |
3/22
PCR (Target)
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
2/25 INPcex colony2 0.1 | VF 1.5 ctl, Rv 1.5 | 21.9 | 25 | 50 |
2/25 INPcex colony2 0.1 | ctl.Fw 1.5 ,VR 1.5 | 21.9 | 25 | 50 |
2/25 INPclos colony1 0.1 | VF 1.5, ctl,Rv 1.5 | 21.9 | 25 | 50 |
2/25 INPclos colony1 0.1 | ctl,Fw 1.5 VR 1.5 | 21.9 | 25 | 50 |
3/22
Miniprep
Notsu, Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
BANcex+RFP col.1 | 147.8 | 1.89 | 2.18 |
BANcex+RFP col.2 | 133.9 | 1.87 | 2.18 |
BANcex+RFP col.1 | 132.4 | 1.85 | 2.19 |
BANclos+RFP col.2 | 126.4 | 1.85 | 2.22 |
INPcex+RFP col.1 | 179.3 | 1.87 | 2.18 |
INPcex+RFP col.2 | 144.9 | 1.87 | 2.19 |
INPclos+RFPcol.1 | 152.2 | 1.87 | 2.23 |
INPclos+RFPcol.2 | 152.4 | 1.86 | |
3/22
PCR (Target)
Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 INP-His9 0.1 | VF 1.5 | ctl.Rv 1.5 | 25 | 21.9 | | 50 |
2/8 INP-His9 0.1 | scFv ctl.Fw 1.5 | VR 1.5 | 25 | 21.9 | | |
3/22
Miniprep
Egashira, Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
3/22 PCR scFv (VF/ctl.RN) | 105.4 | 1.81 | 1.54 |
3/22 PCR scFv (ctl. FV/VR) | 127.4 | 1.87 | |
3/22
OEPCR (Target)
Egashira
Sample1/(µl) | Sample2/(µl) | Primer/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/23 Gel Extraction scfv (VF ctl.Rv)/ 0.1 | 3/23 Gel Extraction scfv (ctl.Fw VR)/ 0.1 | VF/ 1.25, VR/1.25 | | 10.8 | KAPA Taq/ 12.5 | 25 |
3/23
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
5 | 3/22 PCR CBDcex(VF/ctl.Rv) 40 | |
6 | 3/22 PCR CBDcex(ctl.Fw/VR) 40 | |
7 | 3/22 PCR CBDclos(VF/ctl.Rv) 40 | |
8 | 3/22 PCR CBDclos(ctl.Fw/VR) 40 | |
3/23
PCR (Steps)
Kim
Steps | Temparature | Time | Cycle |
Pre Denature | 95℃ | 2min | 1 |
Denature | 98℃ | 20sec | 35 |
Annealing | 60℃ | 15sec | 35 |
Elongation | 72℃ | 15sec | 35 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 10℃ | - | - |
3/23
PCR (Target)
Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/18 Gelex BANcex Fw+VR 0.1, 3?18 Gelex J23105 VF+cexRv 0.1 | VF1.25 | VR 1.25 | 12.5 | 9.8 | | 25 |
3/18 Gelex BANclos closFV VR 0.1, 3/18Gelex J23105 VF+closR 0.1 | VF 1.25 | VR 1.25 | 12.5 | 9.8 | | 25 |
3/23
Gel Extraction (Measurement)
Egashira
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/22 PCR CBDcex(ctlFw/VR) | 26.9 | 1.75 | 1.04 |
3/22PCR CBDclos(ctl Fw/VR) | 100.6 | 1.90 | 1.65 |
3/23
Electrophoresis
Kim
Lane | sample/(µl) | Length(bp) |
3 | 3/18 OEPCR BANcex cexFw+VR J23105 VF+cexRv 25 | |
4 | BANclos clos Fw+VR J23105 VF+clos 25 | |
1 | 1kb ladder 10 | |
2 | 100bp ladder 10 | |
3/23
Electrophoresis
Egashira
Lane | sample/(µl) | Length(bp) |
4 | 3/23 OEPCR CBDcex ctl 25 | |
5 | 3/23 OEPCR CBDclos ctl 25 | |
3/23
PCR (Target)
Egashira
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
3/23 Gel Ex scFv(VF/ctl.Rv) 0.1, 3/23 Gel Ex CBDcex(ctl.Fw/VR) 0.1 | VF 0.75 VR 0.75 | 10.8 | 12.5 | 25 |
3/23 Gel Ex scFv (VF/ctl.Rv), 3/23 Gel Ex clos(ctl.Fw/VR) | VF 0.75 VR 0.75 | 10.8 | 12.5 | 25 |
3/23
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder 5 | |
2 | 100b ladder 7.5 | |
3 | 3/22 PCR scFv(VP/ctl.Rv) 40 | |
4 | 3/22 PCR scFv(ctl.Fw/VR) | |
![](https://static.igem.org/mediawiki/2016/c/c5/T--Kyoto--3_23_No199_Electrophoresis_Koide_09_19_03_50_56%2C1.jpg)
3/24
PCR (Steps)
Kim
Steps | Temparature | Time | Cycle |
Pre Denature | 95℃ | 2min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Elongation | 72℃ | 15sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 10℃ | - | - |
3/24
PCR (Target)
Kim
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/18 Gelcex BAN cex cexFv VR 0.2, 3/18 Gelex J23105 VF cexFv 0.2 | VF1.25 | VR 1.25 | 12.5 | 9.6 | | 25 |
3/18 Gelcex BAN clos clos Fv VR 0.2, 3/18 Gelex J23105 VF clos Rv 0.2 | VF 1.25 | VR 1.25 | 12.5 | 9.6 | | 25 |
3/24
PCR (Purification)
Egashira
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/24 OEPCR CBDcex ctl | 468.6 | 1.82 | 2.06 |
3/24 OEPCR CBDclos ctl | 321.8 | 1.86 | 0.45 |
3/24
PCR (Purification)
Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
3/24 OEPCR BANcex ctl | 77.2 | 1.81 | 1.40 |
3/24 OEPCR BAN clos ctl | 23.8 | 1.75 | 1.56 |
3/24
PCR (Purification)
Kim
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
3/24 OEPCR BANcex ctl | 77.2 | 1.81 | 1.40 |
3/24 OEPCR BAN clos ctl | 23.8 | 1.75 | 1.56 |
3/24
PCR (Purification)
Egashira
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
3/24 OEPCR scFv ctl | 394.2 | 1.83 | |
3/24
Restriction Enzyme Digestion
Egashira
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/24 OE PCR scFv ctl | 5.1 | 1 | | | 1 | 3 | | 19.9 | 30 |
3/24
Transformation
Egashira
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
PCBTC3+scFv+ctl. | | DH5α | |
3/25
Restriction Enzyme Digestion
Egashira
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/24 OEPCR CBDcex ctl | 4.3 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 20.7 | 30 |
3/24 OEPCR CBDclos ctl | 6.2 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 18.8 | 30 |
3/15 J23100+scFv C3-P2 | 9.7 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15.3 | 30 |
3/25
Restriction Enzyme Digestion
Kim
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
3/24 OEPCR BAN-CBDcex ctl | 10 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15 | 30 |
3/24 OEPCR BAN-CBDclos ctl | 10 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 15 | 30 |
3/25
Ligation
Yamada
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
3/24 EPcut pSB1C3 /8 | 3/24 EPcut BAN-CBDcex.ctl /3.7 | 11.7 | 23.4 |
3/24 EPcut pSB1C3 /7 | 3/24 EPcut BAN-CBDclos.ctl /4.7 | 11.7 | 23.4 |
3/25
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
pSB1C3+BAN-CBDcex.ctl 3/25 ligation | - | 20 | LB(CP) |
pSB1C3+BAN-CBDclos.ctl 3/25 ligation | - | 20 | LB(CP) |
pSB1C3+CBDcex 3/24 ligation | - | 20 | LB(CP) |
pSB1C3+CBDclos 3/24 ligation | - | 20 | LB(CP) |
pSB1C3+scFv.ctl 3/24 ligation | - | 20 | LB(CP) |
3/28
Colony PCR (typeA)
Yamada
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
BANcex ctl col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BAncex ctl col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BANclos ctl col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
BANclos ctl col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDcex col.1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDcex col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDclos col,1 | VF 1.25 VR 1.25 | 10 | 12.5 | 25 |
CBDclos col.2 | VF 1.25 VR 1.25 | 10 | 12.5 | |
scFv ctl col.1 | | | | |
scFv ctl col.2 | | | | |
3/28
Electrophoresis
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder(10) | |
2 | 100bb ladder(10) | |
3 | BANcex ctl col.1 | |
4 | BANcex ctl col.2 | |
5 | BANclos ctl col.1 | |
6 | BANclos ctl col.2 | |
7 | CBDcex col.1 | |
8 | CBDcex col.2 | |
9 | CBDclos col.1 | |
10 | CBDclos col.2 | |
11 | scFv ctl col.1 | |
12 | scFv ctl col.2 | |
3/1
Electrophoresis
Li, Kim
Lane | sample/(µl) | Length(bp) |
1 | 100bp ladder /6 | |
2,3 | J23114 /50 | 193+α |
4,5 | J23100 /50 | 193+α |
6,7 | J23108 /50 | 193+α |
3/1
PCR (Purification)
Yamamoto, Kim
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
2/29 PCR J23114 | 4.7 | 1.59 | 0.01 |
2/29 PCR J23100 | 2.4 | 2.60 | 0.01 |
2/29 PCR J23108 | 3.8 | 4.51 | 0.01 |
3/1
PCR (Target)
Yamada
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
3/1 PCR purification J23100 /5 | VF /0.75 | J23100Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
3/1 PCR purification J23108 /5 | VF /0.75 | J23108Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
3/1 PCR purification J23114 /5 | VF /0.75 | J23114Rv /0.75 | - | 6 | Prime Star Premix /12.5 | 25 |
2/22 miniprep m.scFv colony3 /0.5 | BAN+scFv /0.75 | VR /0.75 | - | 10.5 | Prime Star Premix /12.5 | 25 |
7/22
Liquid Culture
Yamamoto
Name | medium |
7/21 transformation BclA-His-scFv colony1 | LB(CP) |
7/21 transformation BclA-His-scFv colony2 | LB(CP) |
7/21 transformation INP-His-scFv colony1 | LB(CP) |
7/21 transformation INP-His-scFv colony2 | LB(CP) |
7/21 transformation BclA-His-CBDcex colony1 | LB(CP) |
7/21 transformation BclA-His-CBDcex colony2 | LB(CP) |
7/21 transformation BclA-His-CBDclos colony1 | LB(CP) |
7/21 transformation BclA-His-CBDclos colony2 | LB(CP) |
7/21 transformation INP-His-CBDcex colony1 | LB(CP) |
7/21 transformation INP-His-CBDcex colony2 | LB(CP) |
7/21 transformation INP-His-CBDclos colony1 | LB(CP) |
7/21 transformation INP-His-CBDclos colony 2 | LB(CP) |
7/25
Miniprep
Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
7/22 liquid culture BclA-His-scFv1 | 183.5 | 1.85 | 2.05 |
7/22 liquid culture BclA-His-scFv2 | 379.0 | 1.86 | 2.20 |
7/22 liquid culture INP-His-scFv1 | 189.6 | 1.85 | 2.21 |
7/22 liquid culture INP-His-scFv2 | 375.3 | 1.85 | 2.21 |
7/22 liquid culture BclA-His-CBDcex1 | 91.9 | 1.87 | 1.90 |
7/22 liquid culture BclA-His-CBDcex2 | 187.1 | 1.87 | 1.98 |
7/22 liquid culture BclA-His-CBDclos1 | 41.2 | 1.88 | 2.21 |
7/22 liquid culture BclA-His-CBDclos2 | 109.4 | 1.94 | 2.24 |
7/22 liquid culture INP-His-CBDcex1 | 71.0 | 1.73 | 1.19 |
7/22 liquid culture INP-His-CBDcex2 | 55.5 | 1.89 | 2.23 |
7/22 liquid culture INP-His-CBDclos1 | 88.6 | 1.82 | 1.61 |
7/22 liquid culture INP-His-CBDclos2 | 75.8 | 1.86 | 2.17 |
7/25
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
2/25 miniprep BclA-His-scFv1 | 10.9 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.1 | 30 |
2/25 miniprep BclA-His-scFv2 | 5.3 | 0 | 1 | 0 | 1 | 10×H /3 | 3.0 | 16.7 | 30 |
2/25 miniprep INP-His-scFv1 | 10.5 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.5 | 30 |
2/25 miniprep BclA-His-CBDcex2 | 10.7 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 14.3 | 30 |
2/25 miniprep BclA-His-CBDclos2 | 18.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 6.7 | 30 |
2/25 miniprep INP-His-CBDcex1 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
2/25 miniprep INP-His-CBDclos1 | 22.6 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 2.4 | 30 |
Samples of "2/25 miniprep BclA-His-scFv1" and "2/25 miniprep INP-His-scFv1" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡis restriction enzyme).
7/26
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 7/25 restriction enzyme digestion BclA-His-scFv1 DraⅡcut | 30 |
3 | 7/25 restriction enzyme digestion BclA-His-scFv2 XPcut | 30 |
4 | 7/25 restriction enzyme digestion INP-His-scFv1 DraⅡcut | 30 |
5 | 7/25 restriction enzyme digestion BclA-His-CBDcex2 SPcut | 30 |
6 | 7/25 restriction enzyme digestion BclA-His-CBDclos2 SPcut | 30 |
7 | 7/25 restriction enzyme digestion INP-His-CBDcex1 SPcut | 30 |
8 | 7/25 restriction enzyme digestion INP-His-CBDclos1 SPcut | 30 |
![](https://static.igem.org/mediawiki/2016/a/af/T--Kyoto--7_26_No96_Gel_Extraction_Notsu_10_02_08_39_23.1.jpg
)
7/26
Gel Extraction(Measurement)
Yamamoto
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
7/26 Gel Extraction BclA-His-scFv2 XPcut | 3.8 | 5.14 | 0.01 |
7/26 Gel Extraction BclA-His-CBDcex2 SPcut | 10.4 | 3.24 | 0.01 |
7/26 Gel Extraction BclA-His-CBDclos2 SPcut | 6.0 | 2.67 | 0.01 |
7/26 Gel Extraction INP-His-CBDcex1 SPcut | 8.1 | 2.03 | 0.02 |
7/26 Gel Extraction INP-His-CBDclos1 SPcut | 14.6 | 2.57 | 0.02 |
7/26
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
7/25 miniprep BclA-His-scFv1 | 10.9 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.1 | 30 |
7/25 miniprep BclA-His-scFv1 | 3.6 | 1 | 0 | 0 | 1 | 10×H /3 | 0 | 3.4 | 10 |
7/25 miniprep BclA-His-scFv1 | 3.6 | 0 | 0 | 0 | 0 | 0 | 0 | 6.4 | 10 |
7/25 miniprep BclA-His-scFv2 | 5.3 | 0 | 1 | 0 | 1 | 10×H /3 | 3.0 | 16.7 | 30 |
/25 miniprep INP-His-scFv1 | 10.5 | 0 | 0 | 0 | 0 | 10×L /3 | 0 | 14.5 | 30 |
/25 miniprep INP-His-scFv1 | 3.5 | 0 | 0 | 0 | 1 | 10×H /1 | 0 | 3.5 | 10 |
/25 miniprep INP-His-scFv1 | 3.5 | 0 | 0 | 0 | 0 | 0 | 0 | 6.5 | 10 |
7/25 miniprep BclA-His-CBDcex2 | 10.7 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 14.3 | 30 |
7/25 miniprep BclA-His-CBDclos2 | 18.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 6.7 | 30 |
7/25 miniprep INP-His-CBDcex2 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
7/25 miniprep INP-His-CBDclos2 | 25 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 0 | 30 |
Samples of "2/25 miniprep BclA-His-scFv(10.9µl)" and "2/25 miniprep INP-His-scFv1(10.5µl)" include 2µl of DraⅡ(Eco0109Ⅰ) (DraⅡis restriction enzyme).
7/26
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 DraⅡcut | 30 |
3 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 XPcut | 10 |
4 | 2/26Restriction Enzyme Digestion BclA-His-scFv1 | 10 |
5 | 2/26Restriction Enzyme Digestion INP-His-scFv1 DraⅡcut | 30 |
6 | 2/26Restriction Enzyme Digestion INP-His-scFv1EPcut | 10 |
7 | 2/26Restriction Enzyme Digestion INP-His-scFv1 | 10 |
- | - | - |
1' | 1kb ladder | 5 |
2' | 2/26Restriction Enzyme Digestion BclA-His-scFv2 XPcut | 30 |
3' | 2/26Restriction Enzyme Digestion BclA-His-CBDcex2 SPcut | 30 |
4' | 2/26Restriction Enzyme Digestion BclA-His-CBDclos2 SPcut | 30 |
5' | 2/26Restriction Enzyme Digestion INP-His-CBDcex2 SPcut | 30 |
6' | 2/26Restriction Enzyme Digestion INP-His-CBDclos2 SPcut | 30 |
![](https://static.igem.org/mediawiki/2016/5/59/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-1.jpg
)
![](https://static.igem.org/mediawiki/2016/3/36/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.1-2.jpg)
![](https://static.igem.org/mediawiki/2016/a/a1/T--Kyoto--7_26_No99_Gel_Extraction_Notsu_10_02_09_03_49.2-1.jpg
)
7/27
Ligation
Yamamoto
Insert DNA(7/27purification) /µ? | Vector DNA(7/25purification) /µ? | Ligation High /µ? | Total |
- | BclA-scFv1 DraⅡcut /7.5 | 7.5 | 15 |
- | INP-scFv1 DraⅡcut /7.5 | 7.5 | 15 |
BclAscFv2 XPcut /7.16 | BclA-CBDcex2 SPcut /0.34 | 7.5 | 15 |
BclAscFv2 XPcut /3.75 | BclA-CBDclos2 SPcut /3.75 | 7.5 | 15 |
BclAscFv2 XPcut /1.88 | INP-CBDcex2 SPcut /5.62 | 7.5 | 15 |
BclAscFv2 XPcut /5.0 | INP-CBDclos2 SPcut /2.5 | 7.5 | 15 |
7/27
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
BclAscFv1 DraⅡcut | 2 | DH5α /20 | LB(CP) |
INPscFv1 DraⅡcut | 2 | DH5α /20 | LB(CP) |
BclAscFv+BclACBDcex | 2 | DH5α /20 | LB(CP) |
BclAscFv+BclACBDclos | 2 | DH5α /20 | LB(CP) |
BclAscFv+INPCBDcex | 2 | DH5α /20 | LB(CP) |
BclAscfv+INPCBDclos | 2 | DH5α /20 | LB(CP) |
7/27
Gel Extraction(Measurement)
Yamamoto
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
7/27 Gel Extraction BclA-His-scFv1 DraⅡcut | 4.8 | -7.11 | 1.04 |
7/27 Gel Extraction INP-His-scFv1 DraⅡcut | 40.4 | 1.32 | 1.00 |
7/27 Gel Extraction BclA-His-scFv2 XPcut | 9.3 | -4.24 | 0.02 |
7/27 Gel Extraction BclA-His-CBDcex2 SPcut | 174.5 | 1.32 | 0.84 |
7/27 Gel Extraction BclA-His-CBDclos2 SPcut | 9.7 | 5.84 | 0.19 |
7/27 Gel Extraction INP-His-CBDcex2 SPcut | 3.7 | -2.82 | 0.14 |
7/27 Gel Extraction INP-His-CBDclos2 SPcut | 22.6 | 2.51 | 0.21 |
7/28
Restriction Enzyme Digestion
Yamamoto
DNA /µ? | EcoR1 /µ? | Xba1 /µ? | Spe1 /µ? | Pst1 /µ? | DraⅡ /µ? | Buffer /µ? | BSA /µ? | MilliQ /µ? | Total /µ? |
BclAscFv2 /5.3 | 0 | 0 | 0 | 0 | 1 | 10×L /3 | 0 | 20.7 | 30 |
BclAscFv2 /5.3 | 0 | 1 | 0 | 1 | 0 | 10×H /3 | 3 | 16.7 | 30 |
INPscFv2 /5.3 | 0 | 0 | 0 | 0 | 1 | 10×L /3 | 0 | 20.7 | 30 |
BclAcex2 /10.7 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 14.3 | 30 |
BclAclos1 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
INPcex1,2 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
INPclos1,2 /25 | 0 | 0 | 1 | 1 | 0 | 10×M /3 | 0 | 0 | 30 |
7/28
Gel Extraction
Kaneko
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
| 7/28 BclA-scFv2 XPcut | |
0.5µg/µl
260/280 2.36
260/230 1.15
7/28
Transformation
Kaneko
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/22 scFv colony2 | 2 | DH5α /20 | |
7/28
Ligation
Yamamoto
Insert DNA /(µl) Vector DNA /(µl) Ligation high /(µl) Total /(µl) |
- 7/28 Restriction Enzyme BclA-scFv DraⅡcut /7.5 7.5 15 |
- 7/28 Restriction Enzyme INP-scFv DraⅡcut /7.5 7.5 1.5 |
7/28
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
7/28 ligation BclA-scFv control | 2 | DH5α /20 | LB(CP) |
7/28 ligation INP-scFv control | 2 | DH5α /20 | LB(CP) |
2/22 miniprep BAN-scFv | | DH5α /20 | |
7/28
Liquid Culture
Yamamoto
Name | medium |
BclA-scFv glycerol stock | LB(CP) |
7/21 transformation INP-scFv colony1 | LB(CP) |
7/21 transformation BclA-CBDcex colony1 | LB(CP) |
7/21 transformation BclA-CBDclos colony1 | LB(CP) |
7/21 transformation INP-CBDcex colony1 | LB(CP) |
7/21 transformation INP-CBDclos colony1 | LB(CP) |
7/21 transformation | LB(CP) |
7/21
Liquid Culture
Yamamoto
Name | medium |
BclA-His-scFv glycerol stock1 | LB(CP) |
BclA-His-scFv glycerol stock2 | LB(CP) |
BclA-His-CBDcex glycerol stock1 | LB(CP) |
BclA-His-CBDcex glycerol stock2 | LB(CP) |
BclA-His-CBDclos glycerol stock1 | LB(CP) |
BclA-His-CBDclos glycerol stock2 | LB(CP) |
INP-His-CBDcex glycerol stock1 | LB(CP) |
INP-His-CBDcex glycerol stock2 | LB(CP) |
INP-His-CBDclos glycerol stock1 | LB(CP) |
INP-His-CBDclos glycerol stock2 | LB(CP) |
7/21
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/8 miniprep INP-His-scFv | 1 | DH5α /20 | LB(CP) |
2/8 miniprep BclA-His-scFv14 | 1 | DH5α /20 | LB(CP) |
2/24 miniprep BclA-His-CBDcex | 1 | DH5α /20 | LB(CP) |
2/24 miniprep BclA-His-CBDclos | 1 | DH5α /20 | LB(CP) |
2/25 miniprep INP-His-CBDcex | 1 | DH5α /20 | LB(CP) |
2/25 miniprep INP-His-CBDclos | 1 | DH5α /20 | LB(CP) |
7/22
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/4 parts awakening E0040 | 1 | DH5α /20 | LB(AMP) |
8/1
Electrophoresis
Iguchi
Lane | sample/(µl) | Length(bp) |
1 | 8/1 colonyPCR BclA control colony4 /10 | 672 |
2 | 8/1 colonyPCR BclA control colony5 /10 | 672 |
3 | 8/1 colonyPCR BclA control colony6 /10 | 672 |
4 | 8/1 colonyPCR INP control colony4 /10 | 1563 |
5 | 8/1 colonyPCR INP control colony5 /10 | 1563 |
6 | 8/1 colonyPCR INP control colony6 /10 | |
7 | 1kb ladder /5 | |
![](https://static.igem.org/mediawiki/2016/8/83/T--Kyoto--8_1_No12_Electrophoresis_Notsu_09_30_08_29_26.1.jpg)
8/1
Restriction Enzyme Digestion
Iguchi
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H 3 | 3 | 13.64 | 30 |
8/1 miniprep BclA-CBDcex colony1 | 18.2 | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
8/1 miniprep BclA-CBDclos colony1 | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
8/1 miniprep INP-CBDcex colony1 | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos colony1 | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | |
8/2
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | BclA-scFv XPcut | 30 |
2 | BclA-CBDcex SPcut | 30 |
3 | BclA-CBDclos SPcut | 30 |
4 | INP-CBDcex SPcut | 30 |
5 | INP-CBDclos SPcut | 30 |
6 | 1kb ladder | 30 |
![](https://static.igem.org/mediawiki/2016/2/28/T--Kyoto--8_2_No14_Gel_Extraction_Notsu_09_30_08_40_44.1.jpg)
8/2
Gel Extraction
Yamamoto
Sample Concentration ng/µl 260/280 260/230 |
8/2 Electrophoresis BclA-scFv XPcut 1.0 23.20 0.12 |
8/2 Electrophoresis BclA-CBDcex SPcut 10.8 1.89 1.35 |
8/2 Electrophoresis BclA-CBDclos SPcut 18.0 1.92 1.88 |
8/2 Electrophoresis INP-CBDcex SPcut 25.4 1.93 1.81 |
8/2 Electrophoresis INP-CBDclos SPcut 60.9 |
8/2
Ligation
Yamamoto
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
8/2 purification BclA-CBDcex SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification BclA-CBDclos SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification INP-CBDcex SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | 15 |
8/2 purification INP-CBDclos SPcut /3.5 | 8/2 purification BclA-scFv XPcut /4 | 7.5 | |
8/2
Transformation
Yamamoto, Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/2 ligation BclA-scFv+BclA-CBDcex | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+BclA-CBDclos | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+INP-CBDcex | 2 | DH5α /20 | LB(CP) |
8/2 ligation BclA-scFv+INP-CBDclos | 2 | DH5α /20 | |
8/2
Liquid Culture
Yamada
Name | medium |
BclA-CBDcex glycerol stock | LB(CP) |
BclA-CBDclos glycerol stock | LB(CP) |
INP-CBDcex glycerol stock | LB(CP) |
INP-CBDclos glycerol stock | |
8/3
Liquid Culture
Yamada
Name | medium |
7/28 transformation BclA control colony5 | LB(CP) |
7/28 transformation BclA control colony6 | LB(CP) |
7/28 transformation INP control colony4 | LB(CP) |
7/28 transformation INP control colony5 | |
7/28 transformation INP control colony6 | |
8/3
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H /3 | 3 | 13.64 | 30 |
8/1 miniprep BclA-CBDcex | 18.2 | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
8/1 miniprep BclA-CBDclos | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
8/1 miniprep INP-CBDcex | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | |
8/3
Ligation
Yamamoto
Vector/(µl) | Insert/(µl) | Ligation High/(µl) | Total/(µl) |
8/1 Restriction Enzyme Digestion BclA-CBDcex /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion BclA-CBDclos /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion INP-CBDcex /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/1 Restriction Enzyme Digestion INP-CBDclos /3.5 | 8/1 Restriction Enzyme Digestion BclA-scFv /4 | 7.5 | 15 |
8/3
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/3 ligation BclA-scFv+BclA-CBDcex | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+BclA-CBDclos | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+INP-CBDcex | 15 | DH5α /20 | LB(CP) |
8/3 ligation BclA-scFv+INP-CBDclos | 15 | DH5α /20 | |
8/4
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
8/3 transformation BclA-scFv+BclA-CBDcex colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDcex colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+BclA-CBDclos colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDcex colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony1 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony2 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony3 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/3 transformation BclA-scFv+INP-CBDclos colony4 | VF 1.25 VR1.25 | 10 | 12.5 | 25 |
8/4
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 1min | 1 |
Denature | 95℃ | 15sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Extension | 72℃ | 10sec | 30 |
Final Elongation | 72℃ | 5min | 1 |
Final Holding | 4℃ | - | - |
8/4
Miniprep
Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/3 liquid culture BclA control colony5 | 137.2 | 1.77 | 1.03 |
8/3 liquid culture BclA control colony6 | 117.6 | 1.87 | 1.54 |
8/3 liquid culture INP control4 | 165.2 | 1.87 | 1.68 |
8/3 liquid culture INP control5 | 150.6 | 1.90 | 1.81 |
8/3 liquid culture INP control6 | 147.4 | 1.79 | |
8/4
Sequence
Yamamoto, Yamada
No primer(10µM) MilliQ Total |
1 / 2 BclA_5_VF(2.18µl) / BclA_5_VR(2.18µl) 2µl / 2µl 5.82µl / 5.82µl 10µl |
3 / 4 BclA_6_VF(2.55µl) / BclA_6_VR(2.55µl) 2µl / 2µl 5.45µl / 5.45µl 10µl |
5 / 6 INP_4_VF(1.81µl) / INP_4_VR(1.81µl) 2µl / 2µl 6.19µl / 6.19µl 10µl |
7 / 8 INP_5_VF(1.99µl) / INP_5_VR(1.99µl) 2µl / 2µl 6.01µl / 6.01µl 10µl |
8/4
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony1 /10 | 1716 |
2 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony2 /10 | 1716 |
3 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony3 /10 | 1716 |
4 | 8/4 colony PCR BclA-scFv+BclA-CBDcex colony4 /10 | 1716 |
5 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony1 /10 | 1716 |
6 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony2 /10 | 1716 |
7 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony3 /10 | 1716 |
8 | 8/4 colony PCR BclA-scFv+BclA-CBDclos colony4 /10 | 1716 |
9 | 100bp ladder /5 | - |
10 | 1kb ladder /5 | - |
11 | - | - |
12 | - | - |
8/4
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony1 /10 | 2606 |
2 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony2 /10 | 2606 |
3 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony3 /10 | 2606 |
4 | 8/4 colony PCR BclA-scFv+INP-CBDcex colony4 /10 | 2606 |
5 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony1 /10 | 2606 |
6 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony2 /10 | 2606 |
7 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony3 /10 | 2606 |
8 | 8/4 colony PCR BclA-scFv+INP-CBDclos colony4 /10 | 2606 |
9 | 100bp ladder /5 | - |
10 | 1kb ladder /5 | |
11 | - | - |
12 | - | - |
8/5
PartsAwaking
Yamamoto
Description | PartName | Backbone | Well | PlateYear | PlateNumber | MilliQ(µl) |
RFP | J23105 | J61002 | 17N | 2016 | 4 | |
8/5
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/5 Parts awakening J23105 | 1µl | DH5α /20 | |
8/9
Restriction Enzyme Digestion
Iguchi, Yamada
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/1 miniprep BclA-scFv | 8.36 | 0 | 1 | 0 | 1 | 10×H /3 | 3 | 13.69 | 30 |
8/1 and 7/25 miniprep BclA-CBDcex | 18. | 0 | 0 | 1 | 1 | 10×H /3 | 0 | 6.8 | 30 |
2/25 miniprep BclA-CBDclos | 21.3 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 3.7 | 30 |
2/25 and 7/25 minniprep INP-CBDcex | 18.0 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 7.0 | 30 |
8/1 miniprep INP-CBDclos | 10.8 | 0 | 0 | 1 | 1 | 10×M /3 | 0 | 16.2 | 30 |
8/9
Liquid Culture
Yamada
Name | medium |
8/1 transformation J23105 | |
8/9
Miniprep
Yamada
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/3 liquid culture BclA-CBDccex | 225.8 | 1.74 | 1.63 |
8/3 liquid culture BclA-CBDclos | 102.3 | 1.80 | 1.66 |
8/3 liquid culture INP-CBDcex | 112.4 | 1.86 | 1.77 |
8/3 liquid culture INP-CBDclos | 42.8 | 1.76 | 0.78 |
8/9
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
8/10
Gel Extraction
Yamada
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
A1 | 1kb ladder | 10 |
A2 | BclA-scFv 8/9XPcut | 30 |
A3 | BclA-CBDcex 8/9 SPcut | 30 |
A4 | BclA-CBDclos 8/9 SPcu | 30 |
- | - | - |
B1 | 1kb ladder | 10 |
B2 | INP-CBDcex 8/9 SPcut | 30 |
B3 | INP-CBDclos 8/9 SPcut | |
![](https://static.igem.org/mediawiki/2016/b/be/T--Kyoto--8_10_No31_Gel_Extraction_Notsu_10_01_11_59_14.1.jpg)
8/10
Gel Extraction(Measurement)
Yamada
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
BclA-scFv 8/9XPcut | 2.6 | 1.13 | 0.58 |
BclA-CBDcex 8/9 SPcut | 6.3 | 1.54 | 0.97 |
BclA-CBDclos 8/9 SPcut | 4.7 | 1.49 | 0.48 |
INP-CBDcex 8/9 SPcut | 4.4 | 1.39 | 0.67 |
INP-CBDclos 8/9 SPcut | 3.5 | 1.29 | |
8/10
Miniprep
Notsu
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/9 liquid culture J23105 (AMP) RFP colony3 | 178.8 | 1.98 | 1.92 |
8/12
Liquid Culture
Iguchi
Name | medium |
8/2 liquid culture INP-CBDclos | LB(CP) |
BclA-scFv glycerol stock 1 | LB(CP) |
BclA-scFv glycerol stock 2 | LB(CP) |
BclA-scFv glycerol stock 3 | LB(CP) |
BclA-scFv glycerol stock 4 | LB(CP) |
8/18
Miniprep
Notsu
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/12 liquid culture INP-CBDclos | 45.3 | 1.82 | 1.31 |
8/12 liquid culture BclA-scFv glycerol stock 1 | 101.6 | 1.59 | 0.71 |
8/12 liquid culture BclA-scFv glycerol stock 2 | 100.7 | 1.67 | 0.78 |
8/12 liquid culture BclA-scFv glycerol stock 3 | 77.8 | 1.60 | 0.70 |
8/12 liquid culture BclA-scFv glycerol stock 4 | 41.0 | 1.87 | 1.27 |
8/20
Liquid Culture
Ohara, Isoda
Name | medium |
BclA-CBDcex | LB(CP) |
BclA-CBDclos | LB(CP) |
INP-CBDcex | LB(CP) |
INP-CBDclos | |
8/21
Liquid Culture
Ohara
Name | medium |
BclA-scFv ①(glisto) | LB(CP) |
BclA-scFv ② | LB(CP) |
BclA-scFv ③ | LB(CP) |
BclA-scFv ④ | |
8/22
Electrophoresis
Sassa
Lane | sample/(µl) | Length(bp) |
8/22 Restraction Ensyme Digestion 1.1kb ladder | 5 | |
8/22 Restraction Ensyme Digestion Bcla-cex | 25 | 2520 |
8/22 Restraction Ensyme Digestion Bcla-clos | 25 | 2520 |
8/22 Restraction Ensyme Digestion INP-cex(s,pcut) | 25 | 3500 |
8/22 Restraction Ensyme Digestion INP-clos | 25 | 3500 |
8/22 Restraction Ensyme Digestion INP-cex | 25 | 2541 |
8/22 Restraction Ensyme Digestion J23105 (AMP) RFP 8/21 old | 25 | 950 |
8/22 Restraction Ensyme Digestion Liquid culture Bcla-scfv glisto 4 | 25 | 932 |
8/22 Restraction Ensyme Digestion Bcla-scfv glisto 2 | 25 | 932 |
100ladder | 5 | |
![](https://static.igem.org/mediawiki/2016/9/9f/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.1.jpg)
![](https://static.igem.org/mediawiki/2016/a/a4/T--Kyoto--8_22_No49_Electrophoresis_Sassa_08_31_11_29_24.2.jpg)
8/22
Restriction Enzyme Digestion
Ohara
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/12 Liquid cultureBclA-scFv glisto ④ | 38 | 0 | 1 | 0 | 1 | 5 | 5 | | 50 |
8/12 Liquid cultureBclA-scFv glisto ② | 38 | 0 | 1 | 0 | 1 | 5 | 5 | 0 | 50 |
8/22
Liquid Culture
Yoshimoto, Ohara, Isoda
Name | medium |
BclA control | LB(CP) |
INP control | |
8/22
Transformation
Ohara
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/9 J23105 RFP | | DH5α /20 | |
8/22
CBD
Yoshimoto
1st | | | |
sample | before | after | wash |
BclAclos+ | 0.208/0.206/0.206 | 0.131/0.135/0.14 | 0.111/0.112/0.109 |
INPclos+ | 0.183/0.186/0.184 | 0.239/0.23/0.22 | 0.158/0.155/0.169 |
8/22
CBD
Yoshimoto
2nd | | | |
sample | before | after | wash |
BclAclos+ | 0.208/0.205/0.204 | 0.078/0.074/0.075 | 0.056/0.056/0.039 |
INPclos+ | 0.183/0.181/0.187 | 0.093/0.097/0.099 | 0.067/0.073/0.069 |
8/23
CBD
Yamada
2nd | | | |
sample | before | after | wash |
BclAcex+ (15min) | 0.247/0.247/0.248 | 0.221/0.224/0.218 | 0.085/0.075/0.073 |
BclAcex+ (30min) | 0.247/0.247/0.248 | 0.243/0.229/0.228 | 0.034/0.048/0.038 |
BclAcex+ (60min) | 0.247/0.247/0.248 | 0.224/0.221/0.217 | 0.065/0.064/0.065 |
BclAcex- (15min) | 0.247/0.247/0.248 | 0.257/0.253/0.258 | |
BclAcex- (30min) | 0.247/0.247/0.248 | 0.236/0.230/0.235 | |
BclAcex- (60min) | 0.247/0.247/0.248 | 0.249/0.248/0.247 | |
cellulose only (60min) | | 0.021/0.021/0.023 | |
8/23
CBD
Yamada
4th | | | |
sample | before | after | wash |
BclAcex+ (2ml tube) | 0.227/0.226/0.225 | 0.108/0.108/0.108 | 0.076/0.074/0.081 |
BclActl+ (2ml tube) | 0.186/0.203/0.189 | 0.082/0.081/0.082 | 0.055/0.046/0.044 |
BclAcex- (2ml tube) | 0.235/0.237/0.233 | 0.261/0.262/0.254 | |
BclAcex+ (1.5ml tube) | 0.237/0.226/0.236 | 0.159/0.157/0.158 | 0.066/0.063/0.065 |
BclActl+ (1.5ml tube) | 0.177/0.173/0.179 | 0.112/0.109/0.113 | 0.080/0.076/0.095 |
BclAcex- (1.5ml tube) | 0.240/0.235/0.238 | 0.255/0.254/0.253 | |
8/24
Liquid Culture
Sassa
Name | medium |
8/5 transormation J2305 | |
8/24
Liquid Culture
Ohara
Name | medium |
8/24 BclA-scFv 1 | LB(CP) |
8/24 BclA-scFv 2 | LB(CP) |
8/24 BclA-scFv 3 | LB(CP) |
8/24 BclA-scFv 4 | |
8/29
Miniprep
Sassa
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
1. 8/23Liquidculture Bcla scFv | 111.6 | 1.98 | 1.82 |
2. 8/23Liquidculture Bcla scFv | 143.1 | 2.00 | 2.03 |
2'. 8/23Liquidculture Bcla scFv | 141.6 | 2.04 | 2.10 |
3. 8/23Liquidculture Bcla scFv | 145.2 | 2.07 | 2.01 |
8/23Liquidculture J23105 | 63.1 | 2.01 | 1.69 |
8/23Liquidculture J23105 | 68.1 | 2.00 | |
8/24
CBD
Isoda
5th | | | |
sample | before | after | wash |
INPcex+ | 0.195/0.191/0.192 | 0.103/0.102/0.109 | 0.059/0.051/0.055 |
INPcex- | 0.190/0.191/0.205 | 0.186/0.187/0.186 | |
INPclos+ | 0.187/0.187/0.188 | 0.121/0.122/0.126 | 0.051/0.058/0.059 |
INPclos- | 0.196/0.195/0.191 | 0.206/0.204/0.202 | |
INPctl+ | 0.172/0.163/0.162 | 0.116/0.177/0.124 | 0.066/0.063/0.062 |
INPctl- | 0.171/0.175/0.165 | 0.167/0.182/0.171 | |
8/24
CBD
Isoda
6th | null | null | null |
sample | before | after | wash |
INPcex+ | 0.184/0.184/0.189 | 0.104/0.106/0.106 | 0.044/0.044/0.056 |
INPcex- | 0.186/0.190/0.184 | 0.182/0.181/0.184 | |
INPclos+ | 0.184/0.189/0.185 | 0.097/0.098/0.099 | 0.037/0.037/0.038 |
INPclos- | 0.187/0.192/0.190 | 0.185/0.184/0.189 | |
INPctl+ | 0.173/0.184/0.180 | 0.095/0.099/0.095 | 0.045/0.046/0.046 |
INPctl- | 0.185/0.184/0.183 | 0.175/0.175/0.175 | |
BclAcex+ | 0.170/0.168/0.174 | 0.099/0.094/0.092 | 0.037/0.032/0.036 |
BclAcex- | 0.186/0.189/0.171 | 0.164/0.167/0.164 | |
cellulose only | 0.006/0.005/0.006 | 0.013/0.010/0.006 | |
8/25
CBD
Yamada
7th | null | null | null |
sample | before | after | wash |
BclAcex+ | 0.181/0.181/0.181 | 0.192/0.196/0.195 | 0/0/0 |
BclAcex- | 0.149/0.150/0.151 | 0.221/0.213/0.211 | |
BclActl+ | 0.169/0.165/0.167 | 0.154/0.156/0.157 | 0.012/0.013/0.011 |
BclActl- | 0.151/0.153/0.154 | 0.187/0.178/0.173 | |
8/26
PCR (Target)
Sassa
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
8/10 miniprep J23105 /3 | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/24 OD0.206 INP-CBDclos /3 | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/24 OD0.202 INP-CBDcex | VF /1.25 | VR /1.25 | - | 7 | KAPA2GFAST /12.5 | 25 |
8/26
Electrophoresis
Yamamoto, Sassa
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder /5 | |
2 | 8/26 PCR INP-CBDcex VFVR /15 | |
3 | 8/26 PCR INP-CBDclos VFVR /15 | |
8/26
Miniprep
Sassa
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/26 Gel Extraction Bcla cex | 8.3 | 1.52 | 0.25 |
8/26 Gel Extraction Bcla clos | 10.2 | 1.70 | 0.26 |
8/26 Gel Extraction INP cex | 11.4 | 1.76 | 0.15 |
8/26 Gel Extraction INP clos | 7.7 | 1.62 | 0.09 |
8/26 Gel Extraction Bcla scFv glisto | 7.0 | 1.59 | 0.20 |
8/26 Gel Extraction Bcla scFv glisto | 14.1 | 6.33 | |
8/26
Miniprep
Yamamoto, Sassa
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
8/26 Gel Extraction Bcla cex | 8.3 | 1.52 | 0.25 |
8/26 Gel Extraction Bcla clos | 10.2 | 1.70 | 0.26 |
8/26 Gel Extraction INP cex | 11.4 | 1.76 | 0.15 |
8/26 Gel Extraction INP clos | 7.7 | 1.62 | 0.09 |
8/26 Gel Extraction Bcla scFv glisto | 7.0 | 1.59 | 0.20 |
8/26 Gel Extraction Bcla scFv glisto | 14.1 | 6.33 | |
8/26
CBD
Yoshimoto
8th | null | null | null |
sample | before | after | wash |
BclAcex+ | 0.183/0.185/0.188 | 0.116/0.115/0.114 | 0.086/0.086/0.089 |
BclActl+ | 0.116/0.115/0.114 | 0.096/0.092/0.087 | 0.09/0.079/0.084 |
cellulose only | | 0.037/0.035/0.037 | |
8/26
CBD
Ohara
9th | null | null | null |
sample | before | after | wash |
BclAcex+ | 0.186/0.186/0.184 | 0.170/0.169/0.170 | 0.021/0.024/0.020 |
BclAcex- | 0.154/0.149/0.149 | 0.142/0.143/0.141 | |
BclActl+ | 0.194/0.189/0.189 | 0.167/0.164/0.159 | 0.010/0.009/0.008 |
BclActl- | 0.162/0.168/0.169 | 0.159/0.162/0.165 | |
8/29
Ligation
Sassa
Insert DNA | vector DNA | Ligation High | Total |
8/26 Gel Extraction BclAscFv /3.4 | 8/26 Gel Extraction Bcla CBDcex /4.1 | 7.5 | 15 |
8/26 Gel Extraction BclAscFv /3.1 | 8/26 Gel Extraction Bcla CBD clos /4.4 | 7.5 | 15 |
8/26 Gel Extraction BclAscFvBclAscFv /3.8 | 8/26 Gel Extraction Bcla CBD cex /4.7 | 7.5 | 15 |
8/26 Gel Extraction BclAscFv /2.6 | 8/26 Gel Extraction Bcla CBD clos /4.9 | 7.5 | |
8/29
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
8/29
PCR (Target)
Miyazaki
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
BclA-CBDcex | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
BclA-CBDclos | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
INP-CBDcex | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
INP-CBDclos | VF /1.25 | VR /1.25 | - | 10 | KAPA2GFAST /12.5 | 25 |
8/29
ligation
Miyazaki
Insert DNA | Vector DNA | Ligation High | Total |
BclAscFv XPcut 6.3 | INPCBDcex SPcut 1.2 | 7.5 | 15 |
BclAscFv XPcut 6.4 | INPCBDclos SPcut 1.1 | 7.5 | 15 |
8/29
Miniprep
Sassa, Miyazaki
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
BclA scFv 1 | 8.4 | 1.63 | 0.11 |
BclA scFv 2 | 5.6 | 1.91 | 0.04 |
INP CBDcex | 44.8 | 1.90 | 0.70 |
INP CBDclos | 33.1 | 1.82 | 0.68 |
BclA cex | 26.3 | 1.93 | 0.62 |
BclA clos | 24.4 | 1.93 | 0.66 |
J23105 VF VR | 33.2 | 1.88 | 0.24 |
8/1
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
7/28 transformaton BclA control colony4 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton BclA control colony5 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton BclA control colony6 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony4 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony5 | VF/1.25 VR/1.25 | 10 | 12.5 | 25 |
7/28 transformaton INP control colony6 | VF/1.25 VR/1.25 | 10 | 12.5 | |
8/1
PCR (Steps)
Yamamoto
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 1min | 1 |
Denature | 95℃ | 15sec | 30 |
Annealing | 60℃ | 15sec | 30 |
Extension | 72℃ | 10sec | 30 |
Final Elogation | 72℃ | 5min | 1 |
Final Holding | | - | 1 |
8/1
Miniprep
Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
7/28 liquid culture BclA-scFv | 239.1 | 1.87 | 1.78 |
7/28 liquid culture INP-scFv colony1 | 263.4 | 1.86 | 1.99 |
7/28 liquid culture E0040 colony1 | 98.8 | 1.84 | 2.15 |
7/28 liquid culture BclA-CBDcex colony1 | 109.9 | 1.82 | 1.37 |
7/28 liquid culture BclA-CBDclos colony1 | 94.1 | 1.89 | 1.93 |
7/28 liquid culture INP-CBDcex colony1 | 111.4 | 1.82 | 1.39 |
7/28 liquid culture INP-CBDclos colony1 | 185.1 | 1.81 | |
9/5
Liquid Culture
Isoda
Name | medium |
BclA-CBDcex /20ml | LB(CP) |
BclA-control /20ml | LB(CP) |
9/5
Gel Extraction
Sassa, Miyazaki
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
9/5 electrophoresis 2 | J23105 RFP XPcut backbone | |
9/5 electrophoresis 2 | J23105 RFP XPcut insert | |
9/5 electrophoresis 1 | BclAcex SPcut | |
9/5 electrophoresis 2 | BclAclos SPcut | |
9/5 electrophoresis 3 | INPcex SPcut | |
9/5 electrophoresis 4 | INPclos SPcut | |
9/6
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
Bclascfv+BclaCBDcex colony1 | vf1.25 vr1.25 | 10 | KAPA2GFAST 12.5 | 25 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+BclaCBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+INPCBDcex colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | 〃 |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
Bclascfv+INPCBDclos colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony2 | 〃 | 〃 | 〃 | 〃 |
〃colony3 | 〃 | 〃 | 〃 | onaji |
〃colony4 | 〃 | 〃 | 〃 | 〃 |
9/6
Ligation
Yamamoto
Insert DNA(µl) | Vector DNA/(µl) | Ligation High/(µl) | Total/(µl) |
9/5 Gel Extraction BclA cex SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction BclA clos SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction INP cex SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/5 Gel Extraction INP clos SPcut/7 | 9/5 Gel Extraction J23105 xpcut 36/0.5 | 7.5 | 15 |
9/6
Electrophoresis
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/5 | 0 |
2 | 9/6 PCR scfv+Bclacex colony1/15 | 0 |
3 | 〃2/15 | 0 |
4 | 〃3/15 | 0 |
5 | 〃4/15 | 0 |
6 | 9/6 PCR scfv+Bclaclos colony1 | 0 |
7 | 〃2 | 0 |
8 | 〃3 | 0 |
9 | 〃4 | 0 |
10 | 9/6 PCR scfv+INPcex colony1 | 0 |
11 | 〃2 | 0 |
12 | 〃3 | 0 |
13 | 〃4 | 0 |
14 | 9/6 PCR scfv+INPclos colony1 | 0 |
15 | 〃2 | 0 |
16 | 〃3 | 0 |
17 | 〃4 | |
9/6
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
blotting CC
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
9/6
CBD
Isoda
10th | null | null | null |
sample | before | after | wash |
BclAcex+ | 0.239 | 0.222 | |
BclActl+ | 0.212 | 0.190 | |
BclAcex- | 0.239 | 0.233 | |
BclActl- | 0.221 | 0.210 | |
9/6
CBD
Isoda
11th | null | null | null |
sample | before | after | wash |
BclAcex+ (NaCl) | 0.202 | 0.176 | |
BclAcex- (NaCl) | 0.202 | 0.206 | |
BclAcex- (NaCl) | 0.202 | 0.264 | |
BclAcex+ (PBS buffer) | 0.212 | 0.179 | |
BclAcex- (PBS buffer) | 0.212 | 0.209 | |
BclActl+ (PBS buffer) | 0.211 | 0.172 | |
9/7
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
Bclascfv-BclaCBDcex colony5 | vf12.5 vr12.5 | 10 | KAPA2GFASTHSRM 12.5 | 25 |
〃6 | = | = | = | = |
〃7 | = | = | = | = |
〃8 | = | = | = | = |
〃9 | = | = | = | = |
〃10 | = | = | = | = |
9/5 Bclascfv-BclaCBDclos 5 | = | = | = | = |
〃6 | = | = | = | = |
9/5 Bclascfv-INPCBDcex1 | = | = | = | = |
〃2 | = | = | = | = |
〃3 | = | = | = | = |
〃4 | = | = | = | = |
Bclascfv-INPCBDclos1 | = | = | = | = |
〃2 | = | = | = | = |
〃3 | = | = | = | = |
〃4 | = | = | = | |
9/7
Miniprep
Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
Bcla-CBDcex | 99.7 | 1.94 | 2.08 |
Bcla-CBDclos | 73.5 | 1.96 | 2.15 |
INP-CBDcex | 45.3 | 1.99 | 2.14 |
INP-CBDclos | 49.6 | 1.89 | |
9/7
Restriction Enzyme Digestion
Yamamoto
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/5 miniprep INP-CBDcex | 25 | 0 | 0 | 1 | 1 | 10×M/3 | 0 | 0 | 30 |
8/5 miniprep INP-CBDclos | 25 | 0 | 0 | 1 | 1 | 10×M/3 | 0 | 0 | 30 |
9/13 miniprep BclA-scFv1 | 22 | 0 | 1 | 0 | 1 | 10×H/3 | 3 | 0 | 30 |
9/7
Liquid Culture
Yamamoto
Name | medium |
9/5 Transformation BclascFv-BclaCBDcex colony5 | LB(CP)/5ml |
〃BclascFv-BclaCBDclos colony4 | |
9/7
SEM
Ohara
number | sample | OD600 | VLP(µl) |
1 | BclAscFv 3c5 | 0.990 | 1 |
2 | BclAscFv 1c5 | 1.001 | 1 |
3 | INPscFv 3c5 | 1.062 | 1 |
4 | BclA CTL | 1.053 | 1 |
5 | INP CTL | 0.952 | 1 |
9/7
Liquid Culture
Isoda
Name | medium |
Bcla-CBDcex | LB(CP)/30ml |
Bcla-control | |
9/7
CBD
Isoda
12th | null | null | null |
sample | before | after | wash |
BclAcex+ (PBS buffer) | 0.195/0.192 | 0.188/0.189 | |
BclActl+ (PBS buffer) | 0.198/0.195 | 0.192/0.189 | |
BclAcex+ (CB buffer) | 0.186/0.184 | 0.176/0.173 | |
BclActl+ (CB buffer) | 0.172/0.169 | 0.151/0.155 | |
9/7
CBD
Isoda
13th | null | null | null |
sample | before | after | wash |
BclAcex+ (PBS buffer) (Bemliese) | 0.201/0.201 | 0.201/0.201 | |
BclActl+ (PBS buffer) (Bemliese) | 0.224/0.223 | 0.218/0.215 | |
BclAcex+ (CB buffer) (Bemliese) | 0.266/0.257 | 0.243/0.242 | |
BclActl+ (CB buffer) (Bemliese) | 0.178/0.177 | 0.157/0.149 | |
BclAcex+ (PBS buffer) (Kimwipe) | 0.203/0.201 | 0.200/0.196 | |
BclActl+ (PBS buffer) (Kimwipe) | 0.221/0.220 | 0.224/0.229 | |
BclAcex+ (CB buffer) (Kimwipe) | 0.273/0.266 | 0.244/0.242 | |
BclActl+ (CB buffer) (Kimwipe) | 0.172/0.172 | 0.154/0.154 | |
9/8
Gel Extraction
Yamamoto
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 9/7 restriction enzyme design BclA scFv X/Pcut | 30 |
3 | 9/7 restriction enzyme design INP CBDcex S/Pcut | 30 |
4 | 9/7 restriction enzyme design INP CBDclos S/Pcut | 30 |
5 | 100bp ladder | 5 |
![](https://static.igem.org/mediawiki/2016/a/a8/T--Kyoto--9_8_No88_Gel_Extraction_Miyazaki_10_15_12_18_19.1.jpg)
9/8
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
9/5 TransformationINPcex-scFv colony5 | vf 1.25 vr1.25 | 10 | KAPA2GFAST/12.5 | 25 |
〃colony6 | = | = | = | = |
〃colony7 | = | = | = | = |
〃colony8 | = | = | = | = |
9/5 Transformation INPclos-BclascFv colony1 | = | = | = | = |
〃colony2 | = | = | = | = |
〃colony5 | = | = | = | = |
〃colony6 | = | = | = | |
9/8
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 0.2µl |
4 | Sano VLP 0.1µl |
5 | Sano VLP 0.05µl |
6 | Sano VLP 0.02µl |
7 | Sano VLP 0.05µl |
1'antibody time/buffer 1:3000 6ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1day
transfer 100mA 1h
9/8
Ligation
Yamamoto
Insert/µl | Vector/µl | Ligation High/µl | Total |
9/8 Gel Extraction BclA scFv X/Pcut /6 | 9/8 Gel Extraction INP CBDcex S/Pcut /1.5 | 7.5 | 15 |
9/8 Gel Extraction BclA scFv X/Pcut /6 | 9/8 Gel Extraction INP CBDcex S/Pcut /1.5 | 7.5 | 15 |
9/8
Transformation
Yamamoto, Sassa
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
9/8 Ligation Bclascfv-INPCBDcex | 2 | DH5a/20 | LB(CP) |
〃BclascFv-INPCBDclos | 2 | 〃 | 〃 |
9/7Miniprep BclaCBDcex+8/24 Miniprep J23105 | 1 | 〃 | LB(CP+AMP) |
〃Bcla-CBDclos+8/24 J23105 | 1 | 〃 | 〃 |
9/7 Miniprep INP-CBDcex+8/24 Miniprep J23105 | 1 | 〃 | 〃 |
9/7 Miniprep INP-CBDclos+8/24 Miniprep J23105 | 1 | 〃 | |
9/8
Liquid Culture
Isoda
Name | medium |
Bcla-CBDcex | LB(CP)/30ml |
Bcla-CBDcontrol | |
9/8
CBD
Isoda
14th | null | null | null |
sample | before | after | wash |
CBDcex+ (CB buffer) | 0.241/0.236 | 0.215/0.212 | |
CBDctl+ (CB buffer) | 0.182/0.180 | 0.159/0.159 | |
BclAcex+ (CB buffer + Na2CO3) | 0.235/0.235 | 0.177/0.177 | |
BclActl+ (CB buffer+Na2CO3) | 0.227/0.218 | 0.155/0.156 | |
9/9
Glycerol Stock
Yamamoto
sample /ml | glycerol /ml |
BclA control /0.8 | 0.2 |
BclA control /0.8 | 0.2 |
9/9
Glycerol Stock
Yamamoto
sample /ml | glycerol /ml |
INP control /0.8 | 0.2 |
INP control /0.8 | 0.2 |
9/9
Liquid Culture
Yamamoto
9/9
Colony PCR (typeA)
Iguchi
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INPcex-scFv colony9 | vf1.25 vr1.25 | 8.5 | KAPA2GFAST 12.5 | 25 |
〃colony10 | 〃 | 〃 | 〃 | 〃 |
〃colony11 | 〃 | 〃 | 〃 | 〃 |
9/5 transformation INPclos-scFv colony1 | 〃 | 〃 | 〃 | 〃 |
〃colony5 | 〃 | 〃 | 〃 | |
9/9
Liquid Culture
Miyazaki
9/9
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl |
3 | Sano VLP 1µl |
4 | Sano VLP 0.2µl |
5 | Sano VLP 0.1µl |
6 | Sano VLP 0.05µl |
7 | Sano VLP 0.02µl |
8 | Sano VLP 0.5µl 95℃ without boiling |
9 | only SDS lysis buffer |
1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h
9/9
Miniprep
Miyazaki
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
scFv Bcla/1ul | 271.8 | 2.09 | 2.15 |
VLP/1ul | 1.67 | 1.98 | |
9/9
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
BclaCBDcexJ23105 | vf1.25 vr1.25 | 10 | KAPA2GFAST HSRM withDye/12.5 | 25 |
9/6 Transformation BclaCBDclos J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcex J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcexJ23105 | 〃 | 〃 | 〃 | 〃 |
9/9
Transformation
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/24 miniprep Bcla-his-cex colony2 | 0.1 | DH5a/20ul | |
9/9
Liquid Culture
Iguchi
Name | medium |
9/5 transformation INPcex-BclascFv colony9 | LB(CP)/5ml |
〃INPclos-BclascFv colony1 | |
9/9
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
9/5 transformation Bcla CBDcex J23105 | vf1.0 vr1.0 | 23 | Quick Taq/25 | 50 |
〃BclaCBDclos J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDcex J23105 | 〃 | 〃 | 〃 | 〃 |
〃INPCBDclos J23105 | 〃 | 〃 | 〃 | |
9/12
Restriction Enzyme Digestion
Miyazaki
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
9/9miniprep scFv Bcla lc3 | 29ul | | 1 | 0 | 1 | 10×H/5 | 5 | 9 | |
9/12
Electrophoresis
Miyazaki
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/5 | - |
2 | Bcla CBDcex j23105/15① | 789 1140 |
3 | 〃② | 〃 |
4 | Bcla CBDclos J23105① | 757 1140 |
5 | 〃② | 〃 |
6 | INP CBDcex J23105① | 1664 1140 |
7 | 〃② | 〃 |
8 | INP CBDclos J23105① | 1796 1140 |
9 | 〃② | 〃 |
10 | 100bp ladder | |
9/12
Colony PCR (typeA)
Miyazaki
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
Bcla CBDcex J23105① | vf0.5 vr0.5 | 11.5 | Quick Taq/12.5 | 25 |
〃② | 〃 | 〃 | 〃 | 〃 |
〃③ | 〃 | 〃 | 〃 | 〃 |
〃④ | 〃 | 〃 | 〃 | 〃 |
Bcla CBDclos J23105① | 〃 | 〃 | 〃 | 〃 |
〃② | 〃 | 〃 | 〃 | 〃 |
〃③ | 〃 | 〃 | 〃 | 〃 |
〃④ | 〃 | 〃 | 〃 | |
9/12
Colony PCR (typeA)
Miyazaki
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INP CBDcex J23105 | vf0.5 vr0.5 | 11.5 | Quick Taq12.5 | 25 |
INP〃② | = | = | = | = |
〃③ | = | = | = | = |
〃④ | = | = | = | = |
〃clos① | = | = | = | = |
〃② | = | = | = | = |
〃③ | = | = | = | = |
〃④ | = | = | = | |
9/13
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 1µl |
3 | Sano VLP 1µl |
4 | Sano VLP 0.2µl |
5 | Sano VLP 0.1µl |
6 | Sano VLP 0.05µl |
7 | Sano VLP 0.02µl |
8 | Sano VLP 0.5µl 95℃ without boiling |
9 | only SDS lysis buffer |
![](https://static.igem.org/mediawiki/2016/c/c8/T--Kyoto--9_13_No112_General_Miyazaki_10_17_05_58_13.1.jpg)
1'antibody time/buffer 1:3000 9ml 1h
2'antibody time/buffer 1:10000 10ml 1h
Blocking 1h
transfer 100mA 1h
30ml TBS-T/blocking reagent 1.5g
9/14
Liquid Culture
Isoda
Name | medium |
9/9 transformation BclA-CBDcex | LB(CP) |
8/24 transformation BclA-CBDclos | LB(CP) |
8/24 transformation INP-CBDcex | LB(CP) |
8/20 transformation | LB(CP) |
9/14
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl+INP-scFv 100µl |
3 | Sano VLP 0.5µl+BAN-scFv 100µl |
4 | Sano VLP 0.2µl +BAN-scFv 100µl |
5 | Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl |
6 | Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl |
7 | Sano VLP 0.2µl +BANcontrol100µl |
8 | Sano VLP 0.2µl 95℃ without boiling |
9 | Sano VLP 0.5µl 95℃ without boiling |
10 | Marker |
1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g
9/15
Western Blotting
Ohara, Sassa
lane | sample |
1 | Marker |
2 | Sano VLP 0.5µl+INP-scFv 100µl |
3 | Sano VLP 0.5µl+BAN-scFv 100µl |
4 | Sano VLP 0.2µl +BAN-scFv 100µl |
5 | Sano VLP 0.2µl +BAN-scFv 70µl +BANcontrol30µl |
6 | Sano VLP 0.2µl +BAN-scFv 20µl +BANcontrol80µl |
7 | Sano VLP 0.2µl +BANcontrol100µl |
8 | Sano VLP 0.2µl 95℃ without boiling |
9 | Sano VLP 0.5µl 95℃ without boiling |
10 | Marker |
1'antibody time/buffer 1:1000 10ml / Blocking buffer overnight
2'antibody time/buffer 1:2500 10ml / Blocking buffer 1h
Blocking 1h
transfer 100mA 1h
25ml TBS-T/blocking reagent 1.3g
9/15
Liquid Culture
Sassa
Name | medium |
9/7 BclA scFv 3C5 9/7 2 | LB(CP) /20mL |
9/14 BclA cex (9/9 tranceformation) | LB(CP) /20mL |
9/7 INP scFv 3C5 | LB(CP) /20mL |
9/9 BclA scFv 1c3 | LB(CP) /20mL |
9/7 INP CTL | LB(CP) /20mL |
9/7 BclA CTL 2 | LB(CP) /20mL |
9/15
Liquid Culture
Ohara, Isoda
Name | medium |
BclA CBDclos glycerol stock | LB(CP) |
INP-CBDcex glycerol stock | LB(CP) |
INP-CBDclos glycerol stock | LB(CP) |
9/16
Transformation
Uchino, Sassa
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/20 J23105 RFP 1/100 | 20 | DH5α /20 | LB(CP) |
9/19
Transformation
Sassa, Takimoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/24 BAN-cex | 1 | DH5α /20 | LB(CP) |
8/5 INP-cex | 1 | DH5α /20 | LB(CP) |
2/8 INP HIsR | 1 | DH5α /20 | LB(CP) |
2/19 BAN-clos | 1 | DH5α /20 | LB(CP) |
8/5 INP-clos | 1 | DH5α /20 | LB(CP) |
2/4 E1010 | 1 | DH5α /20 | |
8/18 BclA-scFv | 1 | DH5α /20 | LB(CP) |
9/19
Liquid Culture
Sassa, Takimoto
Name | medium |
9/15 Liquid culture BclA scFv 3C5② | LB(CP) /20 |
9/19 Liquid culture Bcla CTL | LB(CP) /20 |
9/15 Liquid culture INP-scFv 3C5 | LB(CP) /20 |
9/15 Liquid culture INP CTL | |
9/20
Liquid Culture
Sassa
Name | medium |
9/19 liquid culture BclA-scFv 3C5-2 | LB(CP) |
9/19 liquid culture BclA-ctl | LB(CP) |
9/19 liquid culture INP-scFv 3C5 | LB(CP) |
9/19 liquid culture INP-ctl | LB(CP) |
9/20
Western Blotting
Ohara, Sassa
lane | sample |
Marker | M.Marker |
1 | INPNC-6His-scFv 300 µl+VLP3 µl |
2 | INPNC-6His-scFv 300 µl+PBS3 µl |
3 | INPNC-6His-ctl300 µl+VLP3 µl |
4 | BclA-6His-scFv 300 µl+VLP3 µl |
5 | BclA-6His-scFv 300 µl+PBS3 µl |
6 | BclA-6His-ctl 300 µl+VLP3 µl |
7 | VLP0.5 µl |
8 | VLP0.2 µl |
9 | |
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 80 sec
(ECL Prime)
9/20
Transformation
Uchino
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
BclA-clos glycerol stock | - | DH5α /20 | LB(CP) |
INP-clos glycerol stock | - | DH5α /20 | LB(CP) |
9/21
PCR (Steps)
Uchino
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 65℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 3min | 1 |
Final Holding | 8℃ | - | - |
9/21
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 miniprep INP-His9 /0.2 | VF /0.75 | m.scFv Rv /0.75 | - | 10.8 | KAPA HiFi Ready Mix /12.5 | 25 |
2/8 miniprep INP-His9 /0.2 | m.scFv Fw /0.75 | VR /0.75 | - | 10.8 | KAPA HiFi Ready Mix /12.5 | 25 |
VF 10µM VR 10µM
9/21
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1 | 1kb ladder | 5 |
2 | 100bp ladder | 5 |
3 | 9/21 PCR INP-His9-1 | 25 |
4 | 9/21 PCR INP-His9-2 | 25 |
![](https://static.igem.org/mediawiki/2016/5/53/T--Kyoto--9_21_No129_Gel_Extraction_Notsu_10_03_01_11_36.1.jpg)
9/21
Liquid Culture
Ohara, Sassa
Name | medium |
9/19 Liquid culture BAN-scFv 3C5② | LB(CP) |
9/19 Liquid culture BAN-CTL | LB(CP) |
9/19 Liquid culture INP-scFv 3C5 | LB(CP) |
9/19 Liquid culture INP-CTL | LB(CP) |
9/15 BAN- scFv 1C3 | LB(CP) |
9/15 BAN-CBDcex | LB(CP) |
9/21
Western Blotting
Ohara, Sassa
lane | sample |
Marker | M.Marker |
1 | INPNC-6His-scFv 200 µl+VLP2 µl |
2 | INPNC-6His-scFv 200 µl+PBS2µl(NC) |
3 | INPNC-6His-ctl200 µl+VLP2 µl(NC) |
4 | BclA-6His-scFv200 ul(Vector pSB3C5)+VLP2 ul |
5 | BclA-6His-scFv 200 µl+VLP2 µl (NC) |
6 | BclA-6His-scFv(Vector pSB1C3)+VLP2 ul |
7 | BclA-6His-CBDcex 200 ul + VLP2 ul (NC) |
8 | PBS200 ul +VLP2 ul (NC) |
9 | VLP1 ul (PC) |
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 60 sec
(ECL Prime)
9/21
Gel Extraction(Measurement)
Uchino
Sample/(µl) | Concentration(µg/µl) | 260/280 | 260/230 |
9/21 PCR INP-His9-1 | 19.3 | 1.63 | 0.54 |
9/21 PCR INP-His9-2 | 48.6 | 1.82 | 0.24 |
9/21
PCR (Steps)
Uchino
Steps | Temparature | Time | Cycle |
Predenature | 95℃ | 5min | 1 |
Denature | 98℃ | 20sec | 30 |
Annealing | 65℃ | 15sec | 30 |
Extension | 72℃ | 30sec | 30 |
Final Elongation | 72℃ | 3min | 1 |
Final Holding | 8℃ | - | |
9/21
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
9/21 Gel Extract INP-His9-1 /1µl+9/21 Gel Extract INP-His9-2 /0.5µl | VF /0.75 | VR /0.75 | - | 9.5 | KAPA HiFi /12.5 | 25 |
9/22
growth carve
Sassa
sample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h |
BAM scFv ① | -0.048 | -0.047 | -0.048 | 1.245 | -0.317 | -0.308 | -0.149 | | | | |
BAM scFv ② | -0.036 | 1.512 | -0.830 | -0.309 | -0.314 | -0.127 | | | | | |
INP scFv ① | -0.047 | -0.050 | -0.644 | 1.045 | -0.307 | -0.153 | -0.142 | | | | |
INP scFv ② | -0.038 | -0.o25 | -0.186 | -0.280 | 0.000 | -0.289 | | | | | |
BAN CBDcex ① | -0.045 | -0.048 | -0.044 | 1.089 | -0.322 | -0.284 | -0.296 | | | | |
BAN CBDcex ② | -0.034 | 1.204 | -1.057 | -0.311 | 0.268 | -0.285 | | | | | |
BAN CBDclos ① | 0.035 | -0.037 | -0.035 | 1.261 | -0.323 | -0.160 | -0.287 | | | | |
BAN CBDclos ② | -0.018 | 1.306 | -0.050 | -0.282 | -0.128 | -0.293 | | | | | |
INP CBDcex ① | -0.020 | -0.027 | -0.016 | 1.245 | -0.295 | -0.263 | -0.288 | | | | |
INP CBDcex ② | -0.027 | 1.608 | 0.016 | -0.287 | -0.224 | -0.294 | | | | | |
INP CBDclos ① | -0.034 | -0.034 | -0.032 | 1.026 | -0.318 | -0.235 | -0.270 | | | | |
INP CBDclos ② | -0.026 | 1.415 | 0.012 | -0.297 | -0.047 | -0.272 | | | | | |
BAN CTL ① | -0.039 | -0.039 | -0.036 | 1.274 | -0.314 | -0.230 | -0.271 | | | | |
BAN CTL ② | -0.056 | 1.530 | -0.060 | -0.284 | -0.084 | -3.11 | | | | | |
INP CTL ① | -0.042 | -0.043 | -0.042 | 1.134 | -0.235 | -0.181 | -0.296 | | | | |
INP CTL ② | -0.042 | -1.488 | -0.030 | -0.288 | -0.109 | -0.295 | | | | | |
E1010 ① | -0.041 | -0.040 | -0.036 | 1.171 | -0.322 | -0.252 | | | | | |
E1010 ② | -0.030 | 1.563 | -0.061 | -0.267 | 0.286 | -0.267 | | | | | |
blank ①/② | | | | /-0.307 | -0.306/-0.305 | -0.307/-0.271 | 0.306/ | | | | |
9/22
Liquid Culture
Sassa, Miyazaki
Name | medium |
9/21 liquid culture BAN-CBDcex | LB(AMP) |
9/15 liquid culture INP-ctl | LB(AMP) |
9/15 liquid culture INP-CBDcex | LB(AMP) |
9/15 liquid culture INP-CBDclos | LB(AMP) |
9/15 liquid culture BAN-scFv 3C5 | LB(AMP) |
9/15 liquid culture INP-scFv 3C5 | LB(AMP) |
9/19 liquid culture BAN-CBDclos | LB(AMP) |
9/15 liquid culture BAN-ctl | LB(AMP) |
9/16 transformation J23105-RFP | LB(AMP) |
9/23
SEM
Miyazaki
number | sample(µl) | VLP(µl) | PBS | centrifugal force |
1 | INP scFv /25 | 2.5 | 0 | ×3000g |
2 | INP CTL /25 | 2.5 | 0 | ×3000g |
3 | BclA scFv /25 | 2.5 | 0 | ×3000g |
4 | BclA CTL /25 | 2.5 | 0 | ×3000g |
5 | BclA scFv /25 | 0 | 2.5 | ×3000g |
6 | INP scFv /25 | 2.5 | 0 | ×200g |
7 | INP CTL /25 | 2.5 | 0 | ×200g |
8 | BclA scFv /25 | 2.5 | 0 | ×200g |
9 | BclA CTL /25 | 2.5 | 0 | ×200g |
10 | BclA scFv /25 | 0 | 2.5 | ×200g |
11 | 0 | 2.5 | 0 | ×200g |
9/23
RNA extraction
Isoda
number | sample name |
1 | BclA-CBD cex |
2 | BclA-CBDclos |
3 | INP-CBDcex |
4 | INP-CBDclos |
5 | BclA-scFv |
6 | INP-scFv |
9/23
growth carve
sample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h | 9h | 10h |
9/19 Liquid culture BAN scFv① | 0.003 | 0.001 | 0.000 | 0.008 | 0.015 | 0.009 | 0.013 | 0.016 | 0.015 | 0.022 | 0.062 | 0.020 | 0.027 |
9/19 Liquid culture BAN scFv② | 0.013 | 0.012 | 0.000 | 0.000 | 0.000 | 0.000 | 0.001 | 0.011 | 0.011 | 0.015 | 0.022 | 0.027 | 0.057 |
9/19 Liquid culture INP scFv① | 0.012 | 0.000 | 0.000 | 0.011 | 0.006 | 0.006 | 0.007 | 0.008 | 0.013 | 0.025 | 0.036 | 0.098 | 0.076 |
9/19 Liquid culture INP scFv② | 0.007 | 0.005 | 0.000 | 0.002 | 0.003 | 0.002 | 0.012 | 0.012 | 0.019 | 0.029 | 0.034 | 0.047 | 0.072 |
9/19 Liquid culture BAN cex① | 0.014 | 0.005 | 0.010 | 0.008 | 0.011 | 0.009 | 0.014 | 0.014 | 0.016 | 0.029 | 0.038 | 0.043 | 0.083 |
9/19 Liquid culture BAN cex② | 0.011 | 0.011 | 0.010 | 0.008 | 0.004 | 0.004 | 0.020 | 0.021 | 0.036 | 0.047 | 0.065 | 0.087 | 0.109 |
9/19 Liquid culture BAN clos① | 0.000 | 0.000 | 0.000 | 0.002 | 0.009 | 0.003 | 0.009 | 0.014 | 0.019 | 0.014 | 0.016 | 0.024 | 0.046 |
9/19 Liquid culture BAN clos② | 0.005 | 0.007 | 0.000 | 0.000 | 0.000 | 0.003 | 0.012 | 0.022 | 0.020 | 0.035 | 0.055 | 0.086 | 0.117 |
9/19 Liquid culture INP cex① | 0.187 | 0.000 | 0.001 | 0.007 | 0.011 | 0.006 | 0.013 | 0.016 | 0.020 | 0.023 | 0.037 | 0.048 | 0.068 |
9/19 Liquid culture INP cex② | 0.002 | 0.009 | 0.000 | 0.001 | 0.001 | 0.002 | 0.015 | 0.017 | 0.023 | 0.036 | 0.053 | 0.080 | 0.111 |
9/19 Liquid culture INP clos① | 0.006 | 0.000 | 0.000 | 0.023 | 0.011 | 0.003 | 0.011 | 0.013 | 0.016 | 0.022 | 0.033 | 0.058 | 0.068 |
9/19 Liquid culture INP clos② | 0.029 | 0.028 | 0.000 | 0.009 | 0.000 | 0.016 | 0.001 | 0.007 | 0.008 | 0.014 | 0.020 | 0.029 | 0.074 |
9/19 Liquid culture BAN CTL① | 0.004 | 0.000 | 0.000 | 0.000 | 0.002 | 0.000 | 0.002 | 0.012 | 0.015 | 0.012 | 0.027 | 0.055 | 0.081 |
9/19 Liquid culture BAN CTL② | 0.002 | 0.002 | 0.001 | 0.000 | 0.001 | 0.005 | 0.017 | 0.024 | 0.031 | 0.042 | 0.059 | 0.073 | 0.113 |
9/19 Liquid culture INP CTL① | 0.011 | 0.000 | 0.032 | 0.013 | 0.017 | 0.010 | 0.015 | 0.033 | 0.031 | 0.035 | 0.037 | 0.019 | 0.026 |
9/19 Liquid culture INP CTL② | 0.013 | 0.020 | 0.000 | 0.001 | 0.000 | 0.003 | 0.009 | 0.016 | 0.021 | 0.026 | 0.044 | 0.059 | 0.136 |
9/19 Liquid culture E1010① | 0.015 | 0.016 | 0.014 | 0.011 | 0.016 | 0.018 | 0.032 | 0.066 | 0.108 | 0.173 | 0.258 | 0.361 | 0.393 |
9/19 Liquid culture E1010② | 0.064 | 0.010 | 0.007 | 0.011 | 0.012 | 0.019 | 0.029 | 0.041 | 0.061 | 0.097 | 0.150 | 0.180 | |
9/23
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/5 | |
2 | 9/21 OEPCR/25 | |
9/23
OEPCR
Uchino
sample1 /µl | sample2 /µl | primer Fw /µl | primer Rv /µl | milliQ /µl | KAPA HiFi /µl | total |
9/21 Gel Extract1 /1 | 9/21 Gel Extract2 /0.5 | VF/ 0.75 | VR /0.75 | 9.5 | 12.5 | 25 |
9/23
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/3 | |
2 | 9/23 OEPCR1 /25 | |
3 | 9/23 OEPCR2 /25 | |
9/23
PCR (Target)
Yamamoto
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 miniprep INP-His-scFv9 /0.4 | m.scFv Fw/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | m.scFv Fw/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | VF/1.25 | m.scFv Rv/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
2/8 miniprep INP-His-scFv9 /0.4 | VF/1.25 | m.scFv Rv/1.25 | KAPA2GFAST/12.5 | 9.6 | 0 | 25 |
9/23 Gel Extraction INP-scFv m.PCR/1.5 | VF/1.25 | VR/1.25 | KAPA2GFAST/12.5 | 8.5 | 0 | 25 |
9/26
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/4 | |
2 | 9/23PCR1 /25 | |
3 | 9/23PCR2 /25 | |
4 | 9/23PCR3 /25 | |
5 | 9/23PCR4 /25 | |
6 | 9/23PCR5 /25 | |
![](https://static.igem.org/mediawiki/2016/6/68/T--Kyoto--9_26_No143_Electrophoresis_Miyazaki_10_06_03_46_00.1.jpg)
9/26
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
9/26 electrophoresis lane 4,5 | | 0.357g |
9/26 electrophoresis lane 2,3 | | 0.354g |
9/26
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
9/26 Gel Extract lane4,5 /2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 7 | | 25 |
9/26 Gel Extract lane2,3 /2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 7 | | 25 |
9/26
Liquid Culture
Ohara
Name | medium |
INP-scFv/30ml | LB |
INP-CTL/30ml | LB |
BAN-scFv/30ml | LB |
Ban CTL/30ml | LB |
9/26
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
Bcla-cex 8/5 miniprep | | BL21 | LB(CP) |
Bcla-CTL 8/4 miniprep | | BL21 | LB(CP) |
Bcla-cex 8/5 miniprep | | BL21 | LB(CP) |
Bcla-CTL 8/4 miniprep | | BL21 | LB(CP) |
9/26
RNA extraction
Isoda
number | sample | conc.µg/µl | 260/280 | 260/230 |
4 | BclAcex | 74.73 | 1.94 | 1.61 |
2 | INPcex | 83 | 1.97 | 1.38 |
5 | INPclos | 120.21 | 1.99 | 1.94 |
3 | INPclos | 58.23 | 1.98 | 1.84 |
6 | BclAscFV | 74.76 | 1.96 | 1.84 |
7 | INPscFv | 80.6 | 1.97 | 1.97 |
9/27
Liquid Culture
Yamada
Name | medium |
BclA-cex /250ml | LB(CP) |
BclA-CTL /250ml | LB(CP) |
9/27
growth carve
Sassa, Takimoto
sample | 0h | 0.5h | 1h | 1.5h | 2h | 3h | 4h | 5h | 6h | 7h | 8h |
9/21Liquid culture BAN scFv | 0.113 | 0.170 | 0.266 | 0.408 | 0.599 | 0.893 | 0.164(1.64) | 0.169(1.69) | 0.232(2.32) | 0.234(2.34) | 0.221(2.21) |
9/22Liquid culture BAN cex | 0.109 | 0.147 | 0.219 | 0.374 | 0.515 | 0.873 | 0.146(1.46) | 0.160(1.60) | 0.176(1.76) | 0.231(2.31) | 0.260(2.60) |
9/19Liquid culture BAN clos | 0.097 | 0.109 | 0.184 | 0.338 | 0.486 | 0.744 | 0.130(1.30) | 0.173(1.73) | 0.199(1.99) | 0.203(2.03) | 0.274(2.74) |
9/21Liquid culture BAN CTL | 0.106 | 0.170 | 0.252 | 0.304 | 0.553 | 0.797 | 0.177(1.77) | 0.155(1.55) | 0.216(2.16) | 0.236(2.36) | 0.257(2.57) |
9/21Liquid culture INP scFv | 0.088 | 0.083 | 0.101 | 0.122 | 0.299 | 0.409 | 0.073(0.73) | 0.121(1.21) | 0.140(1.40) | 0.143(1.43) | 0.195(1.95) |
9/15Liquid culture INP cex | 0.102 | 0.116 | 0.145 | 0.207 | 0.320 | 0.626 | 0.170(1.70) | 0.143(1.43) | 0.176(1.76) | 0.188(1.88) | 0.238(2.38) |
9/22Liquid culture INP clos | --- | 0.133 | 0.209 | 0.373 | 0.572 | 0.930 | 0.156(1.56) | 0.201(2.01) | 0.246(2.46) | 0.237(2.37) | 0.293(2.93) |
9/21Liquid culture INP CTL | 0.112 | 0.155 | 0.237 | 0.409 | 0.582 | 0.955 | 0.153(1.53) | 0.209(2.09) | 0.268(2.68) | 0.288(2.88) | 0.274(2.74) |
9/21Liquid culture E1010 | 0.073 | 0.091 | 0.149 | 0.265 | 0.396 | 0.706 | 0.157(1.57) | 0.182(1.82) | 0.222(2.22) | 0.222(2.22) | |
rpm 160
total10mL
CP 2mL/1L
4h~ 10倍希釈
9/27
Western Blotting
Ohara
lane | sample |
Marker | M.Marker(Wako) |
1 | BclA6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) |
2 | BclA6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
3 | INPNC6His-scFv 300 µl+supernatant of NoVLP 3 µl (washed by PBS) |
4 | INPNC6His ctl 300 µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
5 | BclA6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) |
6 | BclA6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) |
7 | INPNC6His-scFv 300 µl+supernatant of NoVLP 3µl (washed by PBST (0.1% tween20)) |
8 | INPNC6His ctl 300 µl+supernatant of NoVLP 3µl (NC) (washed by PBST (0.1% tween20)) |
9 | PBS 300µl+supernatant of NoVLP 3 µl (NC) (washed by PBS) |
Transfer ;60 min 100 mA(CC)
Blocking ;1h
1’antibody ;anti-NoV pAb(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with blocking buffer 1 h
Detection time 60 sec
(ECL Prime)
9/27
Western Blotting
Ohara, Sassa
lane | sample |
Marker | M.Marker(Wako) 5µl |
1 | BBa_E1010(NC) |
2 | BclA-6His-CBDclos (17 kDa) |
3 | INPNC-6His-CBDclos(47 kDa) |
4 | BclA-6His-CBDcex (17 kDa) |
5 | INPNC-6His-CBDcex (47 kDa) |
6 | BclA-6His-scFv (33 kDa) |
7 | INPNC-6His-scFv (63 kDa) |
8 | BBa_K875004 (PC) Lpp-OmpA-scFv-6His (43kDa) |
Marker | M.Marker(Wako) 3µl |
9 | BBa_K875004 (PC) Lpp-OmpA-scFv-6His(43 kDa) |
Used membrane fraction from 100ml culture at logarithmic growth phase.
Transfer ;60min 100mA(CC)
Blocking ;1h
1’antibody ;anti6His(mouse)
1:1000 with blocking buffer O/N
2’antibody ;anti-mouse HRP
1:2500 with TBST 1h
Detection time 380sec
(ECL Prime)
9/27
Reverse Transcription
Yamada
number | Template/µl | gDNA wipeout buffer/µl | RNAase free water/µl | total/µl |
A1 | BclAcex/12 | 2 | 0 | 14 |
A2 | BclAclos/12 | 2 | 0 | 14 |
A3 | INPcex/8.32 | 2 | 3.68 | 14 |
A4 | INPclos/12 | 2 | 0 | 14 |
A5 | BclAscFv/12 | 2 | 0 | 14 |
A6 | INPscFv/12 | 2 | 0 | 14 |
B1 | BclAcex/12 | 2 | 0 | 14 |
B2 | BclAclos/12 | 2 | 0 | 14 |
B3 | INPcex/8.32 | 2 | 3.68 | 14 |
B4 | INPclos/12 | 2 | 0 | 14 |
B5 | BclAscFv/12 | 2 | 0 | 14 |
B6 | INPscFv/12 | 2 | 0 | 14 |
9/27
Reverse Transcription
Yamada
number | template/µl | RTase/µl | RNAase free water/µl | RT buffer/µl | Primer Mix/µl | Total |
A1 | BclAcex/14 | 1 | 0 | 4 | 1 | 20 |
A2 | BclAclos/14 | 1 | 0 | 4 | 1 | 20 |
A3 | INPcex/14 | 1 | 0 | 4 | 1 | 20 |
A4 | INPclos/14 | 1 | 0 | 4 | 1 | 20 |
A5 | BclAscFv/14 | 1 | 0 | 4 | 1 | 20 |
A6 | INPscFv/12 | 1 | 0 | 4 | 1 | 20 |
B1 | BclAcex/14 | 0 | 1 | 4 | 1 | 20 |
B2 | BclAclos/14 | 0 | 1 | 4 | 1 | 20 |
B3 | INPcex/14 | 0 | 1 | 4 | 1 | 20 |
B4 | INPclos/14 | 0 | 1 | 4 | 1 | 20 |
B5 | BclAscFv/14 | 0 | 1 | 4 | 1 | 20 |
B6 | INPscFv/12 | 0 | 1 | 4 | 1 | 20 |
9/27
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/5 | |
2 | 9/26 PCR INP- His scFv/25 | |
3 | 9/26 PCR INP- His scFv/25 | |
![](https://static.igem.org/mediawiki/2016/e/ed/T--Kyoto--9_27_No152_Electrophoresis_Miyazaki_10_06_04_10_12.1.jpg)
9/27
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
9/27 electrophoresis lane2,3 | | 0.7660g |
nanodrop
17.8ng/nl
260/280 1.79
260/230 0.31
9/27
CBD
Yamada
15th | null | null | null |
sample | before | after | wash |
BclAcex+ (CB buffer) | 0.181/0.212/0.204 | 0.254/0.255/0.254 | 0/0/0 |
BclAcex- (CB buffer) | 0.208/0.275/0.228 | 0.160/0.171/0.162 | |
BclActl+ (CB buffer) | 0.178/0.176/0.171 | 0.082/0.085/0.082 | 0.022/0.02/0.02 |
BclActl- (CB buffer) | 0.218/0.214/0.216 | 0.161/0.160/0.159 | |
BclAcex+ (PBS buffer) | 0.212/0.211/0.210 | 0.333/0.334/0.333 | 0.022/0.022/0.021 |
BclAcex- (PBS buffer) | 0.203/0.202/0.201 | 0.178/0.178/0.180 | |
BclActl+ (PBS buffer) | 0.182/0.181/0.180 | 0.253/0.246/0.246 | 0.021/0.021/0.021 |
BclActl- (PBS buffer) | 0.208/0.205/0.208 | 0.192/0.194/0.194 | |
9/29
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
9/27 Gel Extract/2 | VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 9 | | 25 |
| VF/0.75 | VR/0.75 | 2×KAPA HiFi Hotstart ReadyMix/12.5 | 9 | | 25 |
9/29
Liquid Culture
Ohara
Name | medium |
BAN scFv | LB |
INP clos | LB |
BAN clos | LB |
INP cex | LB |
BAN cex | LB |
K875004 | LB |
9/29
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder/2 | |
2 | 9/29 PCR INP-His-scFv/2 | |
3 | | |
9/29
PCR (Purification)
Uchino
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
9/29 PCR INP-His-scFv | 31.9 | 2.02 | 1.38 |
9/30
Electrophoresis
Uchino, Miyazaki
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | |
2 | BclA clos/4.0 | |
3 | INP clos/5.5 | |
4 | BclA cex/4.5 | |
5 | INP cex/2.5 | |
6 | BclA scFv/4.5 | |
7 | INP scFv/4.0 | |
9/5
Restriction Enzyme Digestion
Miyazaki
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
8/10 J23105 RFP miniprep | 20 | | 1 | 0 | 1 | 10×H 3 | 3 | 2 | |
9/5
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
8/29 Ligation Bclascfv-BclaCBDcex | 2 | CH5a/20 | LB(CP) |
〃Bclascfv-BclaCBDclos | 2 | 〃 | 〃 |
〃Bclascfv-INPCBDcex | 2 | 〃 | 〃 |
〃Bclascfv-INPCBDclos | 2 | 〃 | |
9/5
Electrophoresis
Yamamoto
Lane | sample/(µl) | Length(bp) |
1 | 8/20 SPcut Bcla-CBDcex/25 | 2520 |
2 | 〃Bcla-CBDclos/25 | 〃 |
3 | 〃INP-CBDcex/25 | 〃 |
4 | 〃INP-CBDclos/25 | |
9/5
Electrophoresis
Miyazaki
Lane | sample/(µl) | Length(bp) |
1 | 100bp ladder/5 | 6×loading Dye - |
2 | 8/10 miniprep 9/5 xpcut J23105RFP/30 | |
10/1
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 3RT- target /10 | |
2 | 3RT+ target /10 | |
3 | 3RT- 16s rRNA /10 | |
4 | 3RT+ 16s rRNA /10 | |
5 | 4RT- target /10 | |
6 | 4RT+ target /10 | |
7 | 4RT- 16s rRNA /10 | |
8 | 4RT+ 16s rRNA /10 | |
9 | 100bp ladder /5 | |
10 | A-6 target /10 | |
11 | A-5 target /10 | |
12 | A-6 16s rRNA /10 | |
13 | A-5 16s rRNA /10 | |
14 | B-6 target /10 | |
15 | B-5 target /10 | |
16 | B-6 16s rRNA /10 | |
17 | B-5 16s rRNA /10 | |
10/1
Transformation
Ohara
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
K165002 | | DH5α | |
BclA scFv | | DH5α | |
INP scFv | | DH5α | |
BclA cex | | DH5α | |
INP cex | | DH5α | |
BclA clos | | DH5α | |
INP clos | | DH5α | |
K165002 | | BL21 | |
BclA scFv | | BL21 | |
INP scFv | | BL21 | |
BclA cex | | BL21 | |
INP cex | | BL21 | |
BclA clos | | BL21 | |
INP clos | | BL21 | |
10/4
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
INP CTL 8/4 miniprep | | DH5α/20 | LB(CP) |
10/5
Liquid Culture
Yamada
Name | medium |
INP CTL 10/4 transformation | |
10/5
Western Blotting
Ohara
lane | sample |
Marker | M.Marker(Wako) 5µl |
1 | BBa_E165002(NC) |
2 | BclA-6His-scFv (33 kDa) |
3 | INPNC-6His-scFv (63 kDa) |
4 | BclA-6His-CBDcex (17 kDa) |
5 | INPNC-6His-CBDcex(47kDa) |
6 | BclA-6His-CBDclos (17 kDa) |
7 | INPNC-6His-CBDclos (47 kDa) |
8 | BBa_K875004(PC) Lpp-OmpA-scFv-6His (43 kDa) |
Used pelet from 400 µl overnight culture.
Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP 1:2500 with TBST 1h
Detection time 240 sec
(ECL Prime)
10/6
Liquid Culture
Uchino
Name | medium |
10/5 liquid culture INP CTL /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CTL /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CBD cex(5) /2.5ml | LB(CP) /47.5ml |
10/5 liquid culture INP CBD cex(5) /2.5ml | LB(CP) /47.5ml |
10/6
Liquid Culture
Yamada
Name | medium |
10/5 liquid culture INP CTL /100ml | LB(CP) /100ml |
10/3 liquid culture INP CBD cex /100ml | LB(CP) /200ml |
10/7
CBD
Yamada
16th | null | null | null |
sample | before | after | wash |
INPcex+ | 0.208/0.208/0.211 | 0.224/0.221/0.226 | 0.013/0.012/0.010 |
INPcex- | 0.210/0.216/0.204 | 0.232/0.231/0.232 | |
INPctl+ | 0.196/0.196/0.191 | 0.220/0.217/0.212 | 0.023/0.024/0.025 |
INPctl- | 0.194/0.195/0.196 | 0.207/0.199/0.208 | |
cellulose only | 0.015/0.013/0.015 | 0.031/0.031/0.032 | 0.018/0.017/0.019 |
10/11
Transformation
Yamada
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
2/25 miniprep INPcex | 3 | DH5α /20 | LB(CP)×3 |
2/8 miniprep INP His 12 (INPscFv) | 3 | DH5α /20 | LB(CP)×3 |
2/25 miniprep INPcex | 3 | BL21 /20 | LB(CP)×3 |
2/8 miniprep INP His 12 (INPscFv) | 3 | BL21 /20 | LB(CP)×3 |
10/11
SEM(CBD)
Miyazaki
sample/ml | before | after | wash1 | wash2 |
INP CBDcex 10/1 liquid culture /90 | 0.550 | 0.392 | 0.074/0.084 | -0.004/-0.018 |
INP CBDcex 10/1 liquid culture /45 | 0.550 | 0.432 | 0.002/0.000 | -0.001/0.000 |
10/12
Colony PCR (typeA)
Yamada
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INP-His-scFv-INP-His-CBDcex DH5α colony1 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony2 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony3 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony4 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony6 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony7 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony8 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony9 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony10 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony11 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony12 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony13 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony14 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony15 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony16 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony17 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony18 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony19 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony20 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
10/12
Colony PCR (typeA)
Yamada
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
INP-His-scFv-INP-His-CBDcex DH5α colony21 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
2/8 miniprep INP-His-scFv /0.5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
2/25 miniprep INP-His-CBDcex /0.5 | VF/0.5 VR/0.5 | 11.5 | Quick Taq/12.5 | 25 |
10/12
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony1 /5 | |
2 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony2 /5 | |
3 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony3 /5 | |
4 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony4 /5 | |
5 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony5 /5 | |
6 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony6 /5 | |
7 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony7 /5 | |
8 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony8 /5 | |
9 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony17 /5 | |
10 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony18 /5 | |
11 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony19 /5 | |
12 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony20 /5 | |
13 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony21 /5 | |
14 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony22 /5 | |
15 | INPNC-His-scFv-INPNC-His-CBDcex 10/12 colony PCR colony23 /5 | |
16 | INPNC-His-CBDcex 10/12 colony PCR /5 | |
17 | 1kbp ladder /10 | |
| | |
10/12
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 INP-His 12 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/13
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/8 INP-His 12 /0.1 | VF /0.75 | VR /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/13
Colony PCR
Yamada
sample /µl | primer /µl | KAPA Taq Extra Hotstart /µl | DMSO /µl | MilliQ /µl | total /µl |
INP-cex + INP-scFv colony2 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony7 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony17 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony19 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony9 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony10 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony11 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony12 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony13 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony16 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony23 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony24 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony26 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony27 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony28 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony29 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony30 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony31 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
10/13
Colony PCR
Yamada
sample /µl | primer /µl | KAPA Taq Extra Hotstart /µl | DMSO /µl | MilliQ /µl | total /µl |
INP-cex + INP-scFv colony31 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony32 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony33 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex + INP-scFv colony34 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-scFv /0.25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
INP-cex /0.25 | VF /1.25 VR /1.25 | 12.5 | 0.75 | 9.25 | 25 |
10/13
Miniprep
Uchino
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
10/6 liquid culture INP-His-CBDcex | 27.8 | 1.96 | 1.86 |
10/13
PCR (Purification)
Uchino
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
10/12 PCR INP-His 12 (INP-His coding) | 727.3 | 1.88 | 1.86 |
10/13 PCR INP-His 12 | 1132.1 | 1.89 | 2.04 |
10/13
Restriction Enzyme Digestion
Uchino
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
10/13 PCR purification INP-His coding | 7 | 2 | 0 | 0 | 2 | 10×H /6 | 0 | 44 | 60 |
10/13 PCR purification INP-His 12 | 6 | 0 | 2 | 0 | 2 | 10×M /6 | 0 | 44 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 10×H /6 | 0 | 0 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 0 | 0 | 2 | 2 | 10×H /6 | 0 | 0 | 60 |
10/13
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
1 | INPcex+INPscFv colony2 | |
2 | INPcex+INPscFv colony7 | |
3 | INPcex+INPscFv colony19 | |
4 | INPcex+INPscFv colony9 | |
5 | INPcex+INPscFv colony17 | |
6 | INPcex+INPscFv colony10 | |
7 | INPcex+INPscFv colony11 | |
8 | INPcex+INPscFv colony12 | |
9 | INPcex+INPscFv colony15 | |
10 | INPcex+INPscFv colony16 | |
11 | INPcex+INPscFv colony23 | |
12 | INPcex+INPscFv colony24 | |
13 | INPcex+INPscFv colony25 | |
14 | INPcex+INPscFv colony26 | |
15 | INPscFv /5 | |
16 | INPcex /5 | |
17 | 1kbp ladder | |
10/13
Electrophoresis
Yamada
Lane | sample/(µl) | Length(bp) |
A1 | INPcex+INPscFv colony2 | |
A2 | INPcex+INPscFv colony7 | |
A3 | INPcex+INPscFv colony19 | |
A4 | INPcex+INPscFv colony9 | |
A5 | INPcex+INPscFv colony17 | |
A6 | INPscFv /5 | |
A7 | INPcex /5 | |
A8 | 1kbp ladder /5 | |
B1 | INPcex+INPscFv colony10 | |
B2 | INPcex+INPscFv colony11 | |
B3 | INPcex+INPscFv colony12 | |
B4 | INPcex+INPscFv colony13 | |
B5 | INPcex+INPscFv colony14 | |
B6 | 1kbp ladder /5 | |
10/13
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder | |
2 | 10/13 1A | |
3 | 10/13 2A | |
4 | 10/13 3A | |
5 | 10/13 4A | |
6 | 10/13 5A | |
7 | 10/13 1B | |
8 | 10/13 2B | |
9 | 10/13 3B | |
10 | 10/13 4B | |
11 | 10/15 5B | |
10/13
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
1A+2A | 10/13 PCR purification INP His coding | |
3A~5A | 10/13 midiprep INP His CBDcex | |
1B+2B | 10/13 PCR purification INP His12 | |
3B~5B | 10/13 midiprep INP His CBDcex | |
10/13
Fluorescence Microscopy
Uchino
sample | number | magnification |
INPclos | 13318 | ×200 |
INPclos | 13319~13328 | ×600 |
INP CTL | 13329 | ×200 |
INP CTL | 13330~13338 | ×600 |
cellulose only | 13339 | ×200 |
cellulose only | 13340,13343~13351 | ×600 |
cellulose only | 13341,13342 | ×1000 |
without filter
10/13
Fluorescence Microscopy
Uchino
E.coli | OD600 | cellulose | weight | filter |
- | - | powder | 10 | non-filtered |
INPNC-His-ctl | 0.2 | powder | 10 | non-filtered |
INPNC-His-CBDcex | 0.2 | powder | 10 | non-filtered |
- | - | powder | 10 | filtered |
INPNC-His-ctl | 0.2 | powder | 10 | filtered |
INPNC-His-CBDcex | 0.2 | powder | 10 | filtered |
10/13
Fluorescence Microscopy
Uchino
sample | number | magnification |
INPcex | 13353 | ×200 |
INPcex | 13354~13356,13364,13366 | ×600 |
INPcex | 13357,13365 | ×1000 |
INP CTL | 13367 | ×200 |
INP CTL | 13368~13378 | ×600 |
INP CTL | 13379 | ×1000 |
cellulose only | 13380,13381 | ×200 |
cellulose only | 13382~13391 | ×600 |
cellulose only | | |
with filter
10/13
Fluorescence Microscopy
Uchino
sample | number | magnification |
INPcex | 13392~98 | ×200 |
INPcex | 13399~13408 | ×600 |
INP CTL | 13309~13419 | ×200 |
bemliese | 13420~13430 | ×200 |
with bemliese
10/14
Electrophoresis
Yamamoto
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder /3 | |
2 | 10/13 1A /25 | |
4 | 10/13 2A /25 | |
5 | 10/13 3A /25 | |
6 | 10/13 4A /25 | |
7 | 10/13 5A /25 | |
8 | 10/13 1B /25 | |
9 | 10/13 2B /25 | |
10 | 10/13 3B /25 | |
11 | 10/13 4B /25 | |
12 | 10/13 5B /25 | |
![](https://static.igem.org/mediawiki/2016/b/b9/T--Kyoto--10_14_No186_Electrophoresis_Miyazaki_10_16_04_07_57.1.jpg)
with bemliese
10/14
SEM(CBD)
Miyazaki
sample | membrane shape |
INPcex + cellulose | square |
INP CTL + cellulose | triangle |
cellulose only | pentagon |
10/14
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 10/14 gel extract e2 /5 | |
10/14
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
10/14 electrophoresis 1B longer + 2B longer e1 | 10/13 restriction enzyme design INP His 12 | |
10/14 electrophoresis 1B longer + 2B longer e2 | 10/13 restriction enzyme design INP His12 | |
10/14
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kbp ladder /3 | |
2 | 1A + 2A /3 | |
3 | 3A + 4A + 5A /3 | |
10/14
Gel Extraction
Uchino
Lane | Restriction Enzyme Digestion Product | Volume/(µl) |
10/14 electrophoresis 1B + 2B | 10/13 restriction enzyme design INP His12 | |
10/14 electrophoresis 1A + 2A | 10/13 restriction enzyme design INP His coding | |
10/14 electrophoresis 3A + 4A + 5A | INP His CBDcex | |
10/14
Ligation
Uchino
sample1 1A + 2A /µl | sample2 3A~5A /µl | Ligation high ver.2 | total |
5 | 2.5 | 7.5 | 15 |
3.75 | 3.75 | 7.5 | 15 |
10/14
Transformation
Uchino
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
INP His coding ligationA | 2 | DH5α /20 | LB(CP) |
INP His coding ligationA | 4 | DH5α /20 | LB(CP) |
INP His coding ligationB | 2 | DH5α /20 | LB(CP) |
INP His coding ligationB | 4 | DH5α /20 | LB(CP) |
10/15
PCR (Purification)
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
10/15 INP-His coding i | 207.4 | 1.87 | 2.11 |
10/15 INP-His coding G | 174.7 | 1.88 | 1.09 |
10/15 INP-His coding E | 175.5 | 1.08 | 2.10 |
10/15 INP-His coding M | 212.9 | 1.88 | |
10/15
CBD Binding Assay
Ohara
sample | Buffer | OD |
INP cex | CBC buffer | 1.002 |
simultaneous expression | CBC buffer | 0.994 |
INP CTL | CBC buffer | 1.004 |
INP CTL | PBS | 0.964 |
simultaneous expression | PBS | |
10/15
Restriction Enzyme Digestion
Sample | DNA/(µl) | EcoR1/(µl) | Xba1/(µl) | Spe1/(µl) | Pst1/(µl) | Buffer/(µl) | BSA/(µl) | MilliQ/(µl) | Total/(µl) |
10/15 PCR purification INP His coding i | 29.0 | 2 | 0 | 0 | 2 | 6 | | 21 | 60 |
10/15 PCR purification INP His coding G | 34.3 | 2 | 0 | 0 | 2 | 6 | | 15.7 | 60 |
10/15 PCR purification INP His coding E | 34.2 | 2 | 0 | 0 | 2 | 6 | | 15.8 | 60 |
10/15 PCR purification INP His coding M | 28.2(6000ng) | 2 | 0 | 0 | 2 | 6 | | 21.8 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | | 0 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | | 0 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | 6 | | 0 | 60 |
10/13 midiprep INP-His-CBDcex | 50 | 2 | 0 | 0 | 2 | | | 0 | 60 |
1.5 Dr.Kitabatake
2.6 Agarose S + Promega Gel Extraction
3.7 Agarose S + Dr.Nasuda
4.8 stock
10/15
Gel strecking
Yamada
sample | null |
INPNc-His-scFv | (CP)(LB)plate×3 |
INP-His-CBDcex | |
10/15
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
2/22 INP-His scFv colony3 /0.1 | INP His coding Fw /0.75 | INP His coding Rv /0.75 | 2×KAPA /12.5 | 10.9 | 0 | 25 |
10/15
Colony PCR (typeA)
Yamamoto
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
A1 10/14 INPcex + INPscFv colony1 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A2 10/14 INPcex + INPscFv colony2 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A3 10/14 INPcex + INPscFv colony3 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
A4 10/14 INPcex + INPscFv colony4 | VF/1.25 VR/1.25 | 8.75 | KAPA2G /12.5 DMSO/1.25 | 25 |
10/15
Western Blotting
Ohara
lane | sample |
Marker | M.Marker |
1 | BBa_K875004(PC) Lpp-OmpA-scFv-6His 43kDa |
2 | INPNC-6His-CBDcex 47kDa |
3 | INPNC-6His-scFv 63kDa |
4 | INPNC-6His-CBDcex/INPNC-6His-scFv |
5 | BBa_K165002(NC) |
Used pelet from 500 µl overnight culture, adjested OD600 to 3.1 .
Transfar ;80 min 200 mA(CC)
Blocking ;O/N
1’antibody ;anti-6His(mouse)
1:1000 with blocking buffer 1h
2’antibody ;anti-mouse HRP 1:2500 with TBST 1h
Detection time 900 sec
(ECL Prime)
10/16
Electrophoresis
Yamamoto
Lane | sample/(µl) | Length(bp) |
1kb ladder | | |
10/15 colony PCR colony 1 | 3 | |
10/15 colony PCR colony 2 | 25 | |
10/15 colony PCR colony 3 | 25 | |
10/15 colony PCR colony 4 | | |
10/16
Cellulose binding Assay
Uchino
Cellulose/(mg) | E.coli/(mg) | buffer |
1 | cex/1 | carbonate-bicarbonate buffer |
1 | ctl/1 | carbonate-bicarbonate buffer |
0.5 | cex/1 | carbonate-bicarbonate buffer |
0.5 | ctl/1 | carbonate-bicarbonate buffer |
0.5 | CBC/1 | carbonate-bicarbonate buffer |
0.1 | cex/1 | carbonate-bicarbonate buffer |
0.1 | ctl/1 | |
RT,-3h 500rpm 15:45~21:15 binding
fix with 4×1/100 PFA 10min→centrifuge→PBS→centrifuge
→PBS→centrifuge→PBS→centrifuge→DAP1→centrifuge
→PBS→centrifuge→PBS→through filter→PBS into filter
→centrifuge→PBSinto filter→centrifuge
→suspend with PBS→centrifuge
→suspend with?discoloration inhibitor
10/16
Dual Display Assay for Electron Microscope
E.coli | Cellulose | VLP | buffer |
A.Dual 1mL | 1mg | 10µL | PBS |
B.Dual 1mL | 1mg | | PBS |
C.Dual 1mL | | 10µL | PBS |
D.Dual 1mL | | | PBS |
E.INPctl | 1mg | 10µL | PBS |
F.INPctl | 1mg | | PBS |
G.INPctl | | 10µL | PBS |
H.INPctl | | | PBS |
I | 1mg | | PBS |
J | | 10µL | |
RT,ih incubate,850rpm
Dul=1.020
ctl=1.056
10/16
Electrophoresis
Lane | sample/(µl) | Length(bp) |
1 | 1 kb ladder | 5 |
2 | 10/15 Digestion INP His Coding 2 | 30 /0.94 上0.04下0.10 |
3 | 10/15 Digestion INP His Coding 2 | 30 /0.94 上0.04下0.19 |
4 | 10/13 miniprep INP-His-CBDcex 6 | 30 /0.94 0.11 |
5 | 10/13 miniprep INP-His-CBDcex 6 | 30 /0.94 |
Uchino 16:08~
ABEF→centrifuge→resupend with PBS →centrifuge→through filter→resupend with PBS→through filter→centrifuge→respend with PBS→centrifuge→resupend with PBS→centrifuge→resupend with PBS
IJ Michimori
10/16
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | |
2 | 10/15 colony PCR colony1 | |
3 | 10/15 colony PCR colony2 | |
4 | 10/15 colony PCR colony3 | |
5 | 10/15 colony PCR colony | |
![](https://static.igem.org/mediawiki/2016/e/ef/T--Kyoto--10_16_No8_Electrophoresis_Sassa_10_17_12_16_22.1.jpg)
10/16
PCR (Target)
Sassa
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
2/22 scFv col.3 0.1 | VF 0.75 | VR 0.75 | KAPA HoFi 12.5 | 10.9 | | 25 |
2/22 scFv col.3 0.1 | VF 0.75 | VR 0.75 | KAPA HoFi 12.5 | 10.9 | | |
95℃ 5min
98℃ 20s×35
55℃ 15s ×35
72℃ 30s×35
4℃ infinity
10/16
Miniprep
Uchino, Yamamoto
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
10/15 Gel Extraction INP His coding 長 | 9.6 | 1.88 | 0.27 |
10/15 Gel Extraction INP His coding 短 | 74.4 | 1.87 | 0.84 |
10/15 Gel Extraction INP His CBDcex | 22.0 | 1.97 | 0.33 |
Insert MK | 64.5 | 1.92 | 1.17 |
Vector MK | 56.6 | 2.37 | |
10/16
Ligation
Yamamoto
Insert | Vector | MilliQ | Ligation High |
10/15 Insert MK/2µL | 10/15 Gel Extract INP His CBDcex 5.5µl | 0 | 7.5 |
10/15 Insert MK/1µL | 10/15 Gel Extract INP His CBDcex 6µL | 0 | 7.5 |
10/15 Insert MK/1µL | 10/15 Gel Extract INP His CBDcex 0.5µL | 6 | 7.5 |
10/15 Digestion INP His coding/5µL | 10/15 Digestion INP His CBDcex 2.5µL | 0 | 7.5 |
10/15 Digestion INP His coding/6µL | 10/15 Digestion INP His CBDcex 1.5µL | 0 | 7.5 |
10/15 Digestion INP His coding/4.5µL | 10/15 Digestion INP His CBDcex 3µL | 0 | 7.5 |
10/14Gel Extract INP His coding/5.5 | 10/15 Digestion INP His CBDcex 2µL | 0 | 7.5 |
10/14Gel Extract INP His coding/6 | 10/15 Digestion INP His CBDcex 1.5µL | 0 | 7.5 |
10/15 Digestion INP His coding 3.5µL | 10/15 Digestion INP His CBDcex 4µL | 0 | 7.5 |
10/15 Digestion INP His coding 2.5µL | 10/15 Digestion INP His CBDcex 5µL | 0 | 7.5 |
10/15 Digestion INP His coding 3.75 | 10/15 Digestion INP His CBDcex 3.75 | 0 | |
10/16
Transformation
Yamamoto
Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium |
10/15Ligation Insert MK+Gel Extract1 2µL | 2 | DH5α/2 | |
10/15Ligation Insert MK+Gel Extract2 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion3 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion4 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion5 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion6 2µL | 2 | DH5α/2 | |
10/15Ligation Gel Extract+Digestion7 2µL | 2 | DH5α/2 | |
10/15Ligation Gel Extract+Digestion8 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion9 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion10 2µL | 2 | DH5α/2 | |
10/15Ligation Digestion+Digestion11 2µL | 2 | DH5α/2 | |
10/14 Ligation PuroductA 2µL | 2 | DH5α/2 | |
10/14 Ligation PuroductB 2µL | 2 | | |
10/16
PCR (Purification)
Yamamoto
Sample Name/(µl) | Concentration(ng/µl) | 260/280 | 260/230 |
10/15 PCR scFv INP His codingFw+INP His RV | 284.4 | 1.88 | |
10/16
Liquid Culture
Yamamoto
Name | medium |
10/14 Tranceformation INPHis coding2 colony1 | LB(CP) |
10/14 Tranceformation INPHis coding2 colony2 | |
10/16
non-woven felt
Ohara
sample | null |
cex | 13431×200 |
| 32~41×600 |
ctl | 13431×200 |
10/16
Liquid Culture
Yamamoto
Name | medium |
10/14 Trance formation INP His coding 2 colony1 | LB(CP) |
10/14 Trance formation INP His coding 2 colony2 | |
10/16
fluorescence microscope assay
Uchino
sample | number | magnification |
cex bemliese | 13431 | ×200 |
cex bemliese | 13432~13441 | ×600 |
CTL bemliese | 13442 | ×200 |
CTL bemliese | 13443~13355 | ×600 |
10/16
Colony PCR
Matsumoto, Uchino
number | sample | Any others | Any others(µL) |
1~5 | 10/15Ligation Insert MK+Gel Extract1 | 2×Ready Mix | 12.5 |
6~10 | 10/15Ligation Insert MK+Gel Extract2 | Fwd primer | 1.25 |
11~15 | 10/15Ligation Insert MK+Gel Extract3 | Rev primer | 1.25 |
16~20 | 10/15Ligation Digestion+Digestion4 | MilliQ | 10 |
21~24 | 10/15Ligation Digestion+Digestion5 | | 52×mix |
25~28 | 10/15Ligation Digestion+Digestion6 | 2×Ready | 650 |
29~32 | 10/15Ligation Gel Extract+Digestion7 | Fwd | 65 |
33~36 | 10/15Ligation Gel Extract+Digestion8 | Rev | 65 |
37~40 | 10/15Ligation Digestion+Digestion9 | MilliQ | |
41~44 | 10/15Ligation Digestion+Digestion10 | | |
45~48 | 10/15Ligation Digestion+Digestion11 | | |
×16
16Colony Picking DMSO 0.75 12.75 12
Mix 12.5 212.5 200
Fwd 1.25 21.25 18.75 20
Rev 1.25 21.25 18.75 20
MilliQ 9.25 157.25 138.75 148
16sample colony1~14・・・P15のsample colony
Blue 15・・・2/8 INP-His9
16・・・8/1 miniprep INPcex colony
10/16
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | |
2 | 1 | |
3 | 2 | |
4 | 3 | |
5 | 4 | |
6 | 5 | |
7 | 6 | |
8 | 7 | |
9 | 8 | |
10 | 9 | |
11 | | |
12 | | |
10/16
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | | |
2 | 1kb ladder | |
3 | 11 | |
4 | 12 | |
5 | 13 | |
6 | 14 | |
7 | 15 | |
8 | 16 | |
9 | 17 | |
10 | 18 | |
11 | 19 | |
12 | 20 | |
10/16
Fluorescence Microscopy
Uchino
E.coli | OD600 | cellulose | size / weight | filter |
- | - | bemliese | 5mm×5mm | - |
INPNC-His-ctl | 1 | bemliese | 5mm×5mm | - |
INPNC-His-CBDcex | 1 | bemliese | 5mm×5mm | - |
- | - | powder | 1 | filtered |
INPNC-His-ctl | 1 | powder | 1 | filtered |
INPNC-His-CBDcex | 1 | powder | 1 | filtered |
- | - | powder | 0.5 | filtered |
INPNC-His-ctl | 1 | powder | 0.5 | filtered |
INPNC-His-CBDcex | 1 | powder | 0.5 | filtered |
- | - | powder | 0.1 | filtered |
INPNC-His-ctl | 1 | powder | 0.1 | filtered |
INPNC-His-CBDcex | 1 | powder | 0.1 | filtered |
10/16
fluorescence microscope assay
Uchino
sample | number | magnification |
cex 1mg | 13456 | ×200 |
cex 1mg | 13457~13466 | ×600 |
cex 0.5mg | 13467 | ×200 |
cex 0.5mg | 13468~13378 | ×600 |
cex 0.5mg | 13379 | ×1000 |
CTL 1mg | 13380 | ×200 |
CTL 1mg | 13381~13390 | ×600 |
CTL 0.5mg | 13391 | ×200 |
CTL 0.5mg | 13392~13401 | ×600 |
CTL 0.5mg | 13402 | ×1000 |
cex 0.1mg | 13403 | ×200 |
cex 0.1mg | 13404,13407~13416 | ×600 |
cex 0.1mg | 13405,13406 | ×1000 |
CTL 0.1mg | 13417 | ×200 |
CTL 0.1mg | 13418~13430 | ×600 |
CTL 0.1mg | 13431 | ×200 |
cellulose 0.5mg | 13432~13441 | ×600 |
10/17
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 21 | |
2 | 22 | |
3 | 1kb ladder | |
4 | 23 | |
5 | 24 | |
6 | 25 | |
7 | 26 | |
8 | 27 | |
9 | 28 | |
10 | 29 | |
11 | | |
12 | | |
![](https://static.igem.org/mediawiki/2016/4/48/T--Kyoto--10_17_No22_Electrophoresis_Sassa_10_17_07_25_05.1.jpg)
10/17
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | |
2 | 31 | |
3 | 32 | |
4 | 33 | |
5 | 34 | |
6 | | |
10/17
Electrophoresis
Lane | sample/(µl) | Length(bp) |
1 | 36 | |
2 | 37 | |
3 | 38 | |
4 | 39 | |
5 | | |
6 | | |
10/17
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | 1kb ladder | |
2 | 41 | |
3 | 42 | |
4 | 43 | |
5 | | |
6 | | |
10/17
Electrophoresis
Uchino
Lane | sample/(µl) | Length(bp) |
1 | | |
2 | 1kb ladder | |
3 | 45 | |
4 | 46 | |
5 | 47 | |
6 | | |
10/17
Liquid Culture
Yamamoto
Name | medium |
colony 1,2,3,4,5,11,12,13,14,15,18,19,22,24,26,30 | |
total 16mL
10/17
Miniprep
Uchino
DNA | Concentration/(µg/ml) | 260/280 | 260/230 |
1 | 65.8 | 1.91 | |
2 | 63.2 | 1.90 | 1.84 |
3 | 84.6 | 1.87 | 1.33 |
4 | 100.8 | 1.92 | 1.67 |
5 | 84.6 | 1.89 | 1.59 |
11 | 89.3 | 1.84 | 1.43 |
12 | 87.3 | 1.92 | 1.88 |
13 | 88.0 | 1.89 | 1.59 |
14 | 79.8 | 1.91 | 1.72 |
15 | 83.3 | 1.90 | 1.82 |
18 | 102.5 | 1.88 | 1.50 |
19 | 114.9 | 1.91 | 1.59 |
22 | 109.9 | 1.78 | 1.14 |
24 | 80.4 | 1.93 | 2.00 |
26 | 64.6 | 1.93 | 1.84 |
30 | 88.8 | 1.80 | 1.07 |
total 16mL
10/17
Colony PCR (typeA)
Yamada
Templates/(µl) | Primers/(µl) | MilliQ/(µl) | PreMix/(µl) | Total/(µl) |
10/15gel plated colony 1 | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
10/15gel plated colony | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
INPNC-His-scFv | VF1.25 VR1.25 | 9.25 | KapaTag/12.5 DMSO 0.75 | 25 |
INP-His-CBDcex | VF1.25 VR1.25 | 9.25 | | 25 |
VF 1.25×8=10µL
VR 1.25×8=10µL
Kapa 12.5×8=100µL
DM80 0.75×8=6µL
MilliQ 9.25×8=74µL
Total 200µL
10/17
CBD Binding Assay
Uchino
Dual 1mL OD=1.0 | bemlise 5mm×5mm | VLP Narita104 10µL |
Dual 1mL OD=1.0 | bemlise 5mm×5mm | |
ctll 1mL OD=1.0 | bemlise 5mm×5mm | |
ctl 1mL OD=1.0 | bemlise 5mm×5mm | |
10/1
RNA purification
Ohara
BclAcex | 0.504 |
BclAscFv | 0.788 |
INPscFv | 0.837 |
INPclos | 1.564 |
BclAclos | 0.817 |
INPcex | 0.839 |
10/1
RNA extraction
Uchino
number | sample | conc(ng/µl) | 260/280 | 260/230 | diluted solution volume(200ng/µl) |
1 | INPclos | 359.2 | 2.07 | 1.21 | 11.94 |
2 | BclAcex | 230.9 | 2.03 | 0.85 | 2.31 |
3 | INPcex | 1069.0 | 2.11 | 1.43 | 65.17 |
4 | INPscFv | 320.0 | 2.10 | 0.93 | 9 |
5 | BclAclos | 386.2 | 2.04 | 0.97 | 13.97 |
6 | BclAcex | 441.0 | 2.04 | 0.83 | 18 |
10/1
Reverse Transcription
Uchino
name | copy(high or low) | RT |
3 nega | high copy | - |
3 posi | high copy | + |
4 nega | low copy | - |
4 posi | low copy | + |
10/1
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
10/1 RT×20 3RT- /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
10/1 RT×20 3RT+ /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
10/1 RT×20 4RT- /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
10/1 RT×20 4RT+ /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
9/27 RT×1 A-6 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
9/27 RT×1 A-5 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
9/27 RT×1 B-6 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
9/27 RT×1 B-5 /1.0µl | rRNA16s forward primer1 /0.4µl | rRNA16s reverse primer1 /0.4µl | 2×KAPA HiFi /5µl | 3.2µl | | 10µl |
10/1
PCR (Target)
Uchino
Templates/(µl) | Primer1/(µl) | Primer2/(µl) | Buffer/(µl) | MilliQ/(µl) | Any Other/(µl) | Total/(µl) |
10/1 RT×20 3RT- /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
10/1 RT×20 3RT+ /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
10/1 RT×20 4RT- /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
10/1 RT×20 4RT+ /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
9/27 RT×1 A-6 /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
9/27 RT×1 A-5 /1.0 | INP primer for CBD /0.4 | CBD cex primer Rv /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
9/27 RT×1 B-6 /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |
9/27 RT×1 B-5 /1.0 | scFv primer1 /0.4 | scFv primer2 /0.4 | 2×KAPA HiFi /5 | 3.2 | | 10 |