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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 |