Plasmid Extraction for sg11
Recorder: Dong Yan & Kaiyue Ma
Sample:bacteria containing recombinant plasmid pYeT containing sg11
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
| A260/A280 |
1.89 |
1.86 |
1.76 |
1.88 |
1.87 |
1.81 |
1.86 |
1.88 |
| A260/230 |
2.27 |
2.02 |
1.23 |
2.12 |
2.23 |
1.45 |
1.82 |
2.25 |
| concentration(ng/μL) |
159.3 |
113.5 |
265.7 |
46.8 |
130.5 |
189.3 |
59.7 |
169.3 |
Double digestion of AD, BD and YeUGAP
Recorder: Yinchenguang Lyu
Reaction system:
AD & BD:
4 μL template
1.35 μL EcoRI
2 μL PstI
2 μL 10× FastDigest Buffer
10.65 Sterilized ddH2O
YeUGAP:
5 μL template
1 μL EcoRI
0.35 μL PstI
2 μL 10× FastDigest Buffer
11.65 Sterilized ddH2O
Gel Extraction for the product of the double digestion
Recorder: Yinchenguang Lyu
| sample |
AD |
BD |
YeUGAP |
| A260/A280 |
1.89 |
1.84 |
1.86 |
| A260/230 |
0.48 |
0.85 |
0.79 |
| concentration(ng/μL) |
13.1 |
25.1 |
33.9 |
Recorder: Chenyang Li
Plasmid Extraction for YeWGAP
| sample |
08171001 |
08171002 |
08171003 |
08171004 |
08171005 |
08171006 |
| A260/280 |
1.89 |
1.83 |
1.80 |
1.70 |
2.88 |
1.89 |
| A260/230 |
2.36 |
1.83 |
1.47 |
0.99 |
2.07 |
2.14 |
| concentration(ng/μL) |
916.3 |
359.3 |
300.5 |
193.6 |
53.6 |
132.2 |
Ligation of YeUGAP and AD
Recorder: Yinchenguang Lyu
3 μL YeUGAP
3 μL AD
0.4 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
11.6 μL ddH2O
20 μL in total.
Double digestion of AD,BD and PYU
Recorder:Chengle Zhang
| Sample |
AD(0230) |
BD(0231) |
PYU(0232) |
| A260/A280 |
1.97 |
1.83 |
1.89 |
| A260/A280 |
0.12 |
0.41 |
0.75 |
| concentration(ng/μL) |
24.8 |
62.5 |
105.4 |
Plasmid Extraction for sfGFP1-10
Recorder: Xuefeng Meng
Sample:bacteria containing recombinant plasmid pYeT containing sfGFP1-10
| Number |
1 |
2 |
3 |
4 |
| A260/A280 |
1.93 |
1.89 |
1.92 |
1.93 |
| A260/230 |
2.04 |
1.46 |
1.38 |
2.04 |
| concentration(ng/μL) |
43.2 |
19.7 |
49.3 |
20.8 |
**Recorder：Chenyang Li **
Double Digestion of YeWGAP, ADU, BDW
Procedure:
| number |
YeWGAP18171001 |
YeWGAP18171002 |
YeWGAP18171003 |
YeWGAP18171004 |
ADU1001 |
ADU1002 |
BDW1003 |
BDW1004 |
| sample |
YeWGAP08171001 |
YeWGAP08171002 |
YeWGAP08171003 |
YeWGAP08171004 |
ADU1 |
ADU2 |
BDW3 |
BDW4 |
| nuclease-free water(μL) |
8.4 |
76 |
7.3 |
5.4 |
0 |
4 |
0 |
0 |
| 10*Green Buffer(μL) |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
| plasmid(μL) |
0.6 |
1.4 |
1.7 |
2.6 |
8 |
4 |
8 |
8 |
| EcoRI(μL) |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
0.75 |
| PstI(μL) |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
0.25 |
Mix gently and incubate at 37 degree Celsius for 15 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred),08171001,18171001,18171002,08171003,18171003,18171004,ADU1,ADU1001,ADU2,ADU1002,BDW3,BDW1003,BDW4，BDW1004.)
Ligation of pSB1C3 and AD
Recorder: Yinchenguang Lyu
4 μL pSB1C3
2.8 μL AD
0.4 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
10.8 μL ddH2O
20 μL in total.
DATE:8.18
**Recorder：Chenyang Li **
Double Digestion of YeWGAP,BD
Procedure:
| number |
YeWGAPD8181001 |
YeWGAPD8181006 |
BDD8181001 |
BDD8181002 |
| sample |
YeWGAP08171001 |
YeWGAP08171006 |
BD(336) |
BD(336) |
| nuclease-free water(μL) |
15.6 |
9 |
13.6 |
13.6 |
| 10* Fast Digest Buffer(μL) |
2 |
2 |
2 |
2 |
| plasmid(μL) |
1 |
7.6 |
3 |
3 |
| EcoRI(μL) |
1 |
1 |
1 |
1 |
| PstI(μL) |
0.4 |
0.4 |
0.4 |
0.4 |
20 μL in total.
Mix gently and incubate at 37 degree Celsius for 15 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred),YewGAP08171001,D8181001,YewGAP08171006,D8181006,BD(363),BDD8181001,BDD8181002.)
Plasmid Extraction for YeLGAP LYH
Recorder: Dong Yan
| Number |
1 |
2 |
3 |
4 |
| A260/A280 |
1.84 |
1.87 |
1.91 |
1.87 |
| A260/230 |
2.30 |
2.34 |
2.40 |
2.28 |
| concentration(ng/μL) |
634.4 |
459.6 |
806.8 |
387.5 |
Recorder: Chenyang Li
Gel Extraction of double digestion product of YeWGAP and BD
| sample |
YeWGAPD8181001 |
BDD8181001 |
| A260/A280 |
1.68 |
1.84 |
| A260/A230 |
0.2 |
0.15 |
| concentration(ng/μL) |
19.8 |
21.4 |
| volume(μL) |
50 |
25 |
PCR of AD, BD and sup35
Recorder: Yinchenguang Lyu
| sample |
AD |
BD |
sup35 |
| nuclease-free water(μL) |
22 |
22 |
32.5 |
| 2×HiFi-PCR Master(μL) |
25 |
25 |
0 |
| template(μL) |
1 |
1 |
1 |
| 5×PrimeStar Buffer(μL) |
0 |
0 |
10 |
| dNTP(μL) |
0 |
0 |
4 |
| PrimeStar |
0 |
0 |
0.5 |
| Biobrick Primer-r |
2 |
2 |
1 |
| Biobrick Primer-p |
2 |
2 |
1 |
Purification for the product of the PCR
Recorder: Yinchenguang Lyu
results
| Number |
AD1 |
AD2 |
BD1 |
BD2 |
sup35-1 |
sup35-2 |
sup35-3 |
sup35-4 |
| A260/A280 |
1.83 |
1.83 |
1.85 |
1.86 |
1.80 |
1.72 |
1.84 |
1.78 |
| A260/A230 |
1.83 |
2.20 |
2.10 |
2.18 |
1.05 |
0.85 |
1.08 |
1.27 |
| concentration(ng/μL) |
212.3 |
200.6 |
186.0 |
230.9 |
12.3 |
21.2 |
21.5 |
20.9 |
Ligation of YeWGAP and BD
**Recorder: Chenyang Li **
Procedure:
5 μL YeWGAP
2 μL BD
0.2 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
10.8 μL ddH2O
20 μL in total.
Mix gently and incubate at 22 degree Celsius for an hour.
The number of the product: YBL8181001
Plasmid Extraction for YeWGAP
Recorder: Junjie Zeng and Chenyang Li
Sample:bacteria containing recombinant plasmid YeWGAP
| Number |
YeWGAP08181007 |
YeWGAP08181008 |
YeWGAP08181009 |
YeWGAP08181010 |
| A260/A280 |
1.81 |
1.86 |
1.86 |
1.85 |
| concentration(ng/μL) |
740.5 |
841.2 |
725.0 |
695.9 |
DATE:8.19
**Recorder：Chenyang Li **
Double Digestion of YeWGAP,BD
Procedure:
| number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
| sample |
YeWGAP08171001 |
YeWGAP08171001 |
YeWGAP08171001 |
YeWGAP08171001 |
BD(336) |
BD(336) |
BD(230.9) |
| nuclease-free water(μL) |
11 |
11 |
11 |
11 |
12 |
14.5 |
6.4 |
| 10* Fast Digest Buffer(μL) |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
| plasmid(μL) |
5 |
5 |
5 |
5 |
5 |
3 |
10 |
| EcoRI(μL) |
1.5 |
1.5 |
1.5 |
1.5 |
0.7 |
0.3 |
1.2 |
| PstI(μL) |
0.5 |
0.5 |
0.5 |
0.5 |
0.3 |
0.2 |
0.4 |
20 μL in total.
Mix 1,2,3,4,5,6 gently and incubate at 37 degree Celsius for 45 min.
Mix 7 gently and incubate at 37 degree Celsius for 30 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred),YewGAP08171004,1,2,3,4,5,6,7.)
Plasmid Extraction for YeWGAP
Recorder: Junjie Zeng and Chenyang Li
Sample:bacteria containing recombinant plasmid YeWGAP
| Number |
YeWGAP08181011 |
YeWGAP08181012 |
YeWGAP08181013 |
YeWGAP08181014 |
| A260/A280 |
1.89 |
1.87 |
1.88 |
1.89 |
| concentration(ng/μL) |
818.6 |
416.9 |
807.4 |
900.6 |
Plasmid Extraction for YeLGAP
Recorder: Xuefeng Meng
Sample:bacteria containing plasmid YeLGAP
| Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
| concentration(ng/μL) |
128.1 |
228.7 |
60.5 |
67.0 |
398.3 |
291.6 |
80.8 |
101.1 |
| A260/A280 |
1.83 |
1.73 |
1.93 |
1.93 |
1.86 |
1.82 |
1.90 |
1.86 |
| A260/230 |
2.00 |
1.12 |
2.75 |
2.69 |
2.39 |
1.84 |
2.48 |
1.99 |
Recorder:Weijie Jin and Chenyang Li
Gel Extraction of double digestion product of YeWGAP and BD
| sample |
YeWGAPD8191002 |
BDD8191002 |
| A260/A280 |
1.86 |
1.93 |
| A260/A230 |
0.72 |
0.14 |
| concentration(ng/μL) |
315.1 |
52.5 |
Plasmid Extraction for SBAD-2 4 5
Recorder: Dong Yan
| Number |
-2-1 |
-2-2 |
-2-3 |
-4-1 |
-4-2 |
-4-3 |
-5-1 |
-5-2 |
-5-3 |
| A260/A280 |
1.89 |
1.90 |
1.86 |
1.83 |
1.81 |
1.92 |
1.89 |
1.93 |
1.82 |
| A260/230 |
2.29 |
2.19 |
1.56 |
1.56 |
1.88 |
2.18 |
1.71 |
2.20 |
1.33 |
| concentration(ng/μL) |
77.3 |
72.4 |
61.7 |
120.5 |
77.4 |
57.6 |
81.7 |
79.7 |
68.4 |
The absorption peaks are normal, so the poor concentration may due to lack of bacteria growing time.
Double digestion of PSB1C3
Recorder: Yinchenguang Lyu
6μL PSB1C3
1μL EcoRI
0.35μL PstI
2μL 10×Fast Digest Buffer
13.65μL ddH2O
20μL in total.
result:
Gel Extraction of double digestion product of PSB1C3
Recorder: Yinchenguang Lyu
| sample |
PSB1C3:0802 |
| A260/A280 |
1.86 |
| A260/A230 |
1.28 |
| concentration(ng/μL) |
119.3 |
Ligase of PSB1C3 and BD
Recorder: Yinchenguang Lyu
1μL PSB1C3
4μL BD
2μL 10×T4 DNA Ligase Buffer
0.4μL T4 DNA Ligase
12.6μL ddH2O
DATE:8.20
Ligation of YeWGAP and BD
**Recorder: Chenyang Li **
Procedure:
9 μL YeWGAPD8201002
3.3 μL BDD8201002
0.2 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
5.5 μL ddH2O
20 μL in total.
Mix gently and incubate at 22 degree Celsius for an hour.
The number of the product: YBL8201002 and YBL8201003
Then we do transformation.
**Recorder: Chenyang Li **
PCR of Sup35
Procedure:
9 μL YeWGAPD8201002
3.3 μL BDD8201002
0.2 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
5.5 μL ddH2O
20 μL in total.
Procedure:
1.Prepare 4 PCR tubes and sequentially add：
| sample |
sup35 |
| nuclease-free water(μL) |
30.5 |
| 5×PrimeStar Buffer(μL) |
10 |
| dNTP(μL) |
4 |
| template(μL) |
4 |
| PrimeStar |
0.5 |
| Primer1 |
0.5 |
| Primer2 |
0.5 |
50 μL in total.
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
7 min |
| Duration |
95 |
10s |
| Anneal |
57 |
15s |
| Extend |
72 |
50s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
35 cycles(Duration ~ Extend)
Purification for the product of the PCR of Sup35
Recorder: Chenyang Li
results
| Number |
Sup35P8201001 |
Sup35P8201002 |
Sup35P8201003 |
Sup35P8201004 |
| A260/A280 |
1.87 |
1.88 |
1.86 |
1.87 |
| A260/A230 |
2.29 |
2.29 |
1.91 |
2.13 |
| concentration(ng/μL) |
170.2 |
302.3 |
334.6 |
301.0 |
**Recorder：Chenyang Li **
Double Digestion of YeWGAP,BD
Procedure:
| number |
1 |
2 |
3 |
4 |
| sample |
Sup35P8201002 |
Sup35P8201002 |
Sup35P8201004 |
Sup35P8201004 |
| nuclease-free water(μL) |
4 |
7.8 |
4 |
7.8 |
| 10* Fast Digest Buffer(μL) |
2 |
2 |
2 |
2 |
| plasmid(μL) |
10 |
7 |
10 |
7 |
| EcoRI(μL) |
3 |
2.4 |
3 |
2.4 |
| Bcul(μL) |
2.4 |
0.8 |
2.4 |
0.8 |
20 μL in total.
Mix 1,2,3,4gently and incubate at 37 degree Celsius for 15 min.
result:
(from left to right: Trans 2K plus 2(contain Gelred),1,2,3,4,PSB1C3+AD1,PSB1C3+AD2,PSB1C3+AD3,PSB1C3+AD4,)
DATE:8.22
Ligation of YeWGAP and BD
**Recorder: Chenyang Li **
Procedure:
9 μL YeWGAPD8201002
3.3 μL BDD8201002
0.2 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
5.5 μL ddH2O
20 μL in total.
Mix gently and incubate at 22 degree Celsius for an hour.
The number of the product: YBL8201004.
Then we do transformation.
PCR of sup35
**Recorder: Yinchenguang Lyu **
21μL ddH2O
25μL 2×HiFi PCR Master
2μL template
1μL primer1
1μL primer2
| stage |
temperature |
time |
| step 1 |
95 |
5 min |
| step 2 |
95 |
50 s |
| step 3 |
56 |
1 min |
| step 4 |
72 |
1 min 50 s |
| step 5 |
72 |
10 min |
| step 6 |
4 |
-- |
35 cycles(step 2 ~ step 4)
DATE 8.23
Plasmid Extraction for ligased PSB1C3 and AD
Recorder: Yinchenguang Lyu
results
| sample |
1 |
2 |
3 |
4 |
| A260/A280 |
1.81 |
1.77 |
1.70 |
1.57 |
| A260/A230 |
1.64 |
1.43 |
0.79 |
0.78 |
| concentration(ng/μL) |
175.9 |
204.4 |
216.3 |
346.1 |
Recorder：Yu Xie
Colony picking of plasmid pUC57 containing sfGFP11
We did colony picking of plasmid pUC57 containing sfGFP11. After colony picking, we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.
Recorder: Junjie Zeng & Yu Xie
PCR of sup35
Procedure:
1.Prepare 8 PCR tubes and sequentially add：
| sample |
1,2,3,4,5,6,7,8 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95℃ |
10 min |
| Duration |
95℃ |
1 min |
| Anneal |
58℃ |
1 min |
| Extend |
72℃ |
1 min 50 s |
| Post-Extend |
72℃ |
10 min |
| Final |
4℃ |
-- |
30 cycles(Duration ~ Extend)
Recorder: Yu Xie
Double digestion of sfGFP11
Recorder: Yu Xie
Double digestion of sup35
DATE：8.24
Recorder: Yu Xie
Plasmid Extraction for sfGFP11
Recorder: Yu Xie
PCR of sup35
Procedure:
1.Prepare 8 PCR tubes and sequentially add：
| sample |
1,2,3,4,5,6,7,8 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95℃ |
10 min |
| Duration |
95℃ |
1 min |
| Anneal |
58℃ |
1 min |
| Extend |
72℃ |
1 min 50 s |
| Post-Extend |
72℃ |
10 min |
| Final |
4℃ |
-- |
30 cycles(Duration ~ Extend)
Recorder: Yu Xie
Double digestion of sup35
Recorder：Yu Xie
Ligation of sup35 and pUC57-sfGFP11
Recorder: Yu Xie
Transformation of combinant plasmid pUC57 containing sup35-sfGFP11
Recorder: Yu Xie
PCR of sup35
Procedure:
1.Prepare 8 PCR tubes and sequentially add：
| sample |
1,2,3,4,5,6,7,8 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95℃ |
10 min |
| Duration |
95℃ |
1 min |
| Anneal |
58℃ |
1 min |
| Extend |
72℃ |
1 min 50s |
| Post-Extend |
72℃ |
10 min |
| Final |
4℃ |
-- |
30 cycles(Duration ~ Extend)
Recorder：Yu Xie
Colony picking of sfGFP11
Recorder: Chenyang Li
PCR of pYeWGAP-BD
Procedure:
3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add：
4 μL Sterilized ddH2O,
6.5 μL 2XHiFi Pfu,
0.5 μL primer F,
0.5 μL primer R,
1 μL DNA template.
4.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| step 1 |
95℃ |
10 min |
| step 2 |
95℃ |
30 s |
| step 3 |
58℃ |
45 s |
| step 4 |
72℃ |
45 s |
| step 5 |
72℃ |
10 min |
| step 6 |
4℃ |
-- |
30 cycles(step 2 ~ step 4)
results:
(from left to right: Trans 2K plus 2(contain Gelred), 1, 2, 3, 4)
Plasmid Extraction for ligased YeWGAP and BD
Recorder: Chenyang Li
results
| sample |
YBE8241001 |
YBE8241002 |
YBE8241003 |
YBE8241004 |
| A260/A280 |
1.88 |
1.86 |
1.89 |
1.88 |
| A260/A230 |
2.27 |
1.92 |
2.22 |
2.22 |
| concentration(ng/μL) |
203.1 |
174.7 |
181.3 |
177.8 |
DATE：8.24
Recorder: Chenyang Li
PCR of pYeWGAP-BD
Procedure:
3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add：
3 μL Sterilized ddH2O,
5 μL 2XHiFi Pfu,
0.5 μL primer F,
0.5 μL primer R,
1 μL DNA template.
4.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| step 1 |
95℃ |
10 min |
| step 2 |
95℃ |
30 s |
| step 3 |
58℃ |
45 s |
| step 4 |
72℃ |
45 s |
| step 5 |
72℃ |
10 min |
| step 6 |
4℃ |
-- |
30 cycles(step 2 ~ step 4)
results:
(from left to right: Trans 2K plus 2(contain Gelred),YBE8241001，1，YBE8241002，2，YBE8241003，3，YBE8241004，4，)
DATE:8.25
Recorder: Yu Xie
Plasmid Extraction for sfGFP11
Recorder: Yu Xie
Transformation of combinant plasmid pUC57 containing sup35-sfGFP11
Recorder: Yu Xie
PCR of sup35
Procedure:
1.Prepare 8 PCR tubes and sequentially add：
| sample |
1,2,3,4,5,6,7,8 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95℃ |
10 min |
| Duration |
95℃ |
1 min |
| Anneal |
58℃ |
1 min |
| Extend |
72℃ |
1 min 50 s |
| Post-Extend |
72℃ |
10 min |
| Final |
4℃ |
-- |
30 cycles(Duration ~ Extend)
Recorder：Yu Xie
Colony picking of sup35-sfGFP11
Ligation of YeWGAP and BD
**Recorder: Chenyang Li **
Procedure:
9 μL YeWGAPD8201002
3.3 μL BDD8201002
0.2 μL T4 DNA ligase
2 μL 10×T4 DNA Ligase Buffer
5.5 μL ddH2O
20 μL in total.
Mix gently and incubate at 22 degree Celsius for an hour.
The number of the product: YBL8201005, YBL8201006, YBL8201007, YBL8201008, YBL8201009.
Recorder: Chenyang Li
PCR of pYeWGAP-BD
Procedure:
3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add：
4 μL Sterilized ddH2O,
5 μL 2XHiFi Pfu,
0.5 μL primer F,
0.5 μL primer R,
0.5 μL DNA template.
4.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| step 1 |
95℃ |
10 min |
| step 2 |
95℃ |
30 s |
| step 3 |
58℃ |
45 s |
| step 4 |
72℃ |
45 s |
| step 5 |
72℃ |
10 min |
| step 6 |
4℃ |
-- |
30 cycles(step 2 ~ step 4)
results:
(from left to right: Trans 2K plus 2(contain Gelred),YBE8241001，1，YBE8241002，2，YBE8241003，3，YBE8241004，4，YeWGAP08171001，YeWGAPD8191002，BDD8191002，YBL8201005，Sterilized ddH2O)
Date: 8.26
Recorder: Yu Xie
PCR of sfGFP1-10
Recorder: Yu Xie
Double Digestion of sfGFP1-10
Recorder: Yu Xie
Double Digestion of pSB1C3
Date: 8.27
Recorder: Yu Xie
Ligation of pSB1C3 and sfGFP1-10
Recorder: Yu Xie
Transformation of combinant plasmid pSB1C3 containing sfGFP1-10
Recorder：Yu Xie
Colony picking of combinant plasmid pSB1C3 containing sfGFP1-10
Recorder: Yu Xie
PCR of sfGFP1-10
Recorder: Yu Xie
Double Digestion of sfGFP1-10
Plasmid Extraction for plasmid C3BD35
Recorder: Xuefeng Meng
results
| sample |
1 |
2 |
3 |
| A260/A280 |
2.02 |
1.90 |
1.91 |
| A260/A230 |
1.19 |
1.07 |
1.31 |
| concentration(ng/μL) |
17.0 |
15.5 |
27.9 |
PCR of sfGFP11
Recorder: Xuefeng Meng
results
Date: 8.28
Recorder: Yu Xie
Plasmid Extraction for pSB1C3 containing sfGFP1-10
Recorder: Yu Xie
PCR of bacteria which have recombinant plasmid pSB1C3 containing sfGFP1-10
Procedure:
1.Prepare 4 PCR tubes and sequentially add：
| sample |
1,2,3,4 |
| Sterilized ddH2O |
9 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template(bacteria) |
1.5 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
2.PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
1 min |
| Anneal |
58 |
1 min |
| Extend |
72 |
1 min 50s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Agarose gel electrophoresis Results:
succeed!
Recorder: Yu Xie
PCR of recombinant plasmid pSB1C3 containing sfGFP1-10
Procedure:
- Prepare 4 PCR tubes and sequentially add：
| sample |
1,2,3,4 |
| Sterilized ddH2O |
9.5 μL |
| 2×HiFi-PCR Master |
12.5 μL |
| Template(plasmid) |
1 μL |
| Primer 1 |
1 μL |
| Primer 2 |
1 μL |
| total |
25 μL |
- PCR reaction
Parameters setting：
| stage |
temperature |
time |
| Pre-Duration |
95 |
10 min |
| Duration |
95 |
1 min |
| Anneal |
58 |
1 min |
| Extend |
72 |
1 min 15s |
| Post-Extend |
72 |
10 min |
| Final |
4 |
-- |
30 cycles(Duration ~ Extend)
Agarose gel electrophoresis Results:
succeed!
Date: 8.29
Colony PCR to examine ADW
Recorder: Yinchenguang Lyu
6.25 μL 2×Taq PCR Master Mix
2 μL culture containing Ecoli
0.4 μL bio-p
0.4 μL M13-f
3.45 μL ddH2O
Double Digest to examine ADW and C3AD35
Recorder: Yinchenguang Lyu
result(along with that of the former part)
(from left to right: Trans 2K plus 2(contain Gelred), digested-ADW1, digested-ADW2, C3AD35(1), C3AD35(2), ADW1, ADW2, ADW3, ADW4, ADW5, ADW6)
Date：8.30
Plasmid extraction of pGAL1(site mutation)
Recorder: Xuefeng Meng
| sample |
1 |
2 |
3 |
4 |
| concentratin(ng/μL) |
379.6 |
232.9 |
277.2 |
375.4 |
| A260/A280 |
1.81 |
1.85 |
1.90 |
1.90 |
| A260/A230 |
1.42 |
1.72 |
2.33 |
2.31 |
Designed by 2016 iGEM Team:USTC
Under CC License
Based on Semantic-UI
