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