Team:USTC/Notebook/8 24

Modeling

Everybody

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)