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DATE 8.12

Recorder: Yinchenguang Lyu & Yu Xie PCR of bacteria which have recombinant plasmid pYeT containing sfGFP1-10

Procedure:

  1. Prepare 4 PCR tubes and sequentially add：
sample 1,2,3,4
Sterilized ddH2O 15.75 μL
5×PrimeSTAR Buffer (Mg2+Plus) 5 μL
dNTP Mixture (2.5 mM each) 2 μL
Template(bacteria) 1 μL
Primer 1 0.5 μL
Primer 2 0.5 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl) 0.25 μL
total 25 μL
  1. PCR reaction Parameters setting：
stage temperature time
Pre-Duration 95 10 min
Duration 95 10 s
Anneal 55 15 s
Extend 72 44 s
Post-Extend 72 10 min
Final 4 --

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right:sample 4,3,2,1,marker)

Failed again.

Recorder: Yinchenguang Lyu & Yu Xie PCR of bacteria which have recombinant plasmid pYeT containing sfGFP1-10

Procedure:

  1. Prepare 4 PCR tubes and sequentially add：
sample 1,2,3,4
Sterilized ddH2O 15.75 μL
5×PrimeSTAR Buffer (Mg2+Plus) 5 μL
dNTP Mixture (2.5 mM each) 2 μL
Template(bacteria) 1 μL
Primer 1 0.5 μL
Primer 2 0.5 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl) 0.25 μL
total 25 μL
  1. PCR reaction Parameters setting：
stage temperature time
Pre-Duration 95 10 min
Duration 95 10 s
Anneal 55 15 s
Extend 72 44 s
Post-Extend 72 10 min
Final 4 --

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right:marker, sample 1,2,3,4)

Failed again and again.

Recorder: Yonghao Liang PCR of AD&BD and PFP

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample 1 2 3 4 5 6
SterilizedddH2O 11 μL 11 μL 11 μL 11 μL 11 μL 11 μL
2×HiFi-PCR Master 12.5 μL 0 μL 12.5 μL 0 μL 12.5 μL 0 μL
PrimeSTAR premix 0 μL 12.5 μL 0 μL 12.5 μL 0 μL 12.5 μL
Template (AD) 0.5 μL 0.5 μL 0 μL 0 μL 0 μL 0 μL
Template (BD) 0 μL 0 μL 0.5 μL 0.5 μL 0 μL 0 μL
Template (PFP) 0 μL 0 μL 0 μL 0 μL 0.5 μL 0.5 μL
standard-biobrick-p 0.5 μL 0.5 μL 0.5 μL 0.5 μL 0.5 μL 0.5 μL
standard-biobrick-r 0.5 μL 0.5 μL 0.5 μL 0.5 μL 0.5 μL 0.5 μL
total 25 μL 25 μL 25 μL 25 μL 25 μL 25 μL
  1. PCR reaction

Parameters setting for sample 1,3：

stage temperature time
step 1 95 5 min
step 2 95 1 min
step 3 58 1 min
step 4 72 45 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

Parameters setting for sample 2,4：

stage temperature time
step 1 95 5 min
step 2 95 10 s
step 3 55 15 s
step 4 72 29 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

Parameters setting for sample 5：

stage temperature time
step 1 95 5 min
step 2 95 1 min
step 3 55 1 min
step 4 72 2 min 8 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

Parameters setting for sample 6：

stage temperature time
step 1 95 5 min
step 2 95 10 s
step 3 55 15 s
step 4 72 1 min 11 s
step 5 72 10 min
step 6 4 --

30 cycles(step 2 ~ step 4)

The agarose gel electrophoresis result is as follows: 图片名称

Then we did gel extraction,the parameters of product are as follows:

Number AD BD PFP
A260/A280 1.85 1.87 1.91
A260/A230 0.60 1.41 0.02
concentration(ng/μL) 69.9 73.9 86.7

Plasmid Extraction for sfGFP11 and sup35 Recorder: Ya Jiang and Shuai Shao

Sample:bacteria containing sfGFP11 and sup35

Number 0601 0602 0603 0604 1601 1602 1603 1604
Content sfGFP11 sfGFP11 sup35 sup35 sfGFP11 sfGFP11 sfGFP11 sfGFP11
A260/A280 1.88 1.88 1.90 1.90 1.83 1.89 1.87 1.89
concentration(ng/μL) 535.9 236.0 60.3 17.8 230.9 199.7 663.1 722.3

Plasmid Extraction for AD-PYW and BD-PYW Recorder: Chenyang Li

Sample:bacteria containing AD-PYW and BD-PYW

Number 1001 1002 1003 1004
Content BD-PYW AD-PYW BD-PYW AD-PYW
A260/A280 2.02 1.79 1.79 1.98
concentration(ng/μL) 359.7 115.8 124.0 87.1

Plasmid Extraction for YeLGAP Recorder: Kaiyue Ma

Number 1 2 3 4
A260/A280 1.81 1.87 1.79 1.80
A260/A230 1.08 1.17 1.07 0.80
concentration(ng/μL) 29.0 33.0 63.8 18.3

Plasmid Extraction for sfgfp11 Recorder: Yonghao Liang

Number 1 2 3 4
A260/A280 1.89 1.88 1.88 1.88
A260/A230 2.24 2.18 2.12 2.17
concentration(ng/μL) 416.6 398.5 382.9 429.4

Recorder: Yonghao Liang Transformation of plasmid YeUGAP PS:After we cultivate the bacteria for 10 hours, we take 800 μL of bacteria out from one tube into 400 μL glycerinum. And put all the tubes into fridge. Then we do the plasmid extraction, ,the parameters of product are as follows:

Number 1 2 3 4
A260/A280 1.90 1.67 1.98 1.97
A260/A230 0.94 0.71 1.00 0.99
concentration(ng/μL) 13.1 25.5 9.5 11.1

Recorder: Yonghao Liang Transformation of plasmid YeLGAP

Recorder：Junjie Zeng keep and preserve the bacteria in the glycerin tube at -40℃

We mixed 800μL bacterial suspension(pro+RBS+sfGFP(pSB1C3)) with 50% glycerin in a tube(and numbered them as 0605-1,0605-2,0606-1,0606-2 repectively), then place them in the -20℃ refrigerator.

Recorder: Yu Xie PCR of bacteria which have recombinant plasmid pYeT containing sfGFP1-10

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(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
Post-Extend 72 10 min
Final 4 --

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product

Failed again and again and again.

Recorder: Kaiyue Ma & Yu Xie PCR of bacteria which have recombinant plasmid containing sup35 and sfGFP11

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(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 15 min
Duration 95 50s
Anneal 55 1 min
Extend 72 1min20s
Post-Extend 72 10 min
Final 4 --

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right: marker,control,sample 1,2,3,4)

Recorder: Kaiyue Ma & Yu Xie PCR of bacteria which have recombinant plasmid pSB1A3 containing PRsfGFP1-10

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(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 15 min
Duration 95 50s
Anneal 55 1 min
Extend 72 1min20s
Post-Extend 72 10 min
Final 4 --

30 cycles(Duration ~ Extend)

Agarose gel electrophoresis Results: 图片名称 Agarose gel electrophoresis of PCR product (from left to right: marker,control,sample 1,2,3,4)

Plasmid Extraction for recombinant plasmid pYeT containing sfGFP1-10 Recorder: Weijie Jin & Yu Xie

Results

All Failed.

Recorder：Yu Xie Colony picking of recombinant plasmid pYeT containing sfGFP1-10

We did colony picking of combinant plasmid of pYeT and sfGFP1-10.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

Date: 8.13

Plasmid Extraction for PYescGAP Recorder: Yinchenguang Lyu

Results

sample 1 2 3 4
A260/A280 1.87 1.88 1.88 1.87
A260/A230 1.49 1.23 2.12 2.07
concentration(ng/μL) 143.2 190.8 546.2 459.8

Plasmid Extraction for pro+RBS Recorder: Shuai Shao and Kaiyue Ma

Results

sample 1609 1010 1607 1608
Content pro+RBS pro+RBS pro+RBS pro+RBS
A260/A280 1.84 1.85 1.82 1.85
concentration(ng/μL) 100.3 93.6 83.0 104.8

Plasmid Extraction for sup35+sfGFP11 Recorder: Chenyang Li and Shuai Shao

Results

sample 1003 1004 1607 1608
Content sup35+sfGFP11 sup35+sfGFP11 sup35+sfGFP11 sup35+sfGFP11
A260/A280 1.86 1.86 1.87 1.87
concentration(ng/μL) 229.7 235.5 233.4 241.7

After extraction, we conducted electrolysis to see if we got the desired product, the result of which is shown below. From left to right, the stripes are corresponding to marker marker 2K, 1003, 1004, 1607, 1608 and marker 2K+ and the control plasmid.

Gel Extraction for sfGFP11 Recorder: Shuai Shao

Results

sample 1605 1606
Content sfGFP11 sfGFP11
A260/A280 1.75 1.86
concentration(ng/μL) 10.3 7.5

Purification for PCR products of sfGFP11 Recorder: Wenkai Han

Results

sample 1511 1512
Content sfGFP11 sfGFP11
A260/A280 1.86 1.86
concentration(ng/μL) 215.4 152.8

Enzymatic digestion for purified PCR products of sfGFP11 and plasmid of sfGFP11 Recorder: Wenkai Han, Shuai Shao

Enzymatic digestion system

1.Purified PCR products of sfGFP11

sample volume
Pstl 2ul
Xbal 0.35ul
10x DNA buffer 2ul
PCR product 10 or 9ul
ddH2O 15.65 or 16.65ul
total 30ul

2.Plasmid of sfGFP11

sample volume
Pstl 1ul
Xbal 1ul
10x DNA buffer 2ul
PCR product 1ul
total 20ul

After enzymatic digestion, we conducted electrolysis to see if we got the desired product, the result of which is shown below. This shows that we have got the desired outcome, sfGFP11.

Gel Extraction for pro+RBS Recorder: Weijie Jin

Results

sample 1401
Content pro+RBS
A260/A280 1.93
concentration(ng/μL) 18.7

Plasmid Extraction for recombinant plasmid pYeT containing sfGFP1-10 Recorder: Chengle Zhang & Yu Xie

Results

All Failed.

Recorder: Yu Xie Transformation of recombinant plasmid pYeT containing sfGFP1-10 Bacteria: top 10 Plates: Ap+ 50 μg/mL

Recorder：Yu Xie Colony picking of combinant plasmid pYeT containing sfGFP1-10

We did colony picking of combinant plasmid of pYeT and sfGFP1-10.After colony picking,we cultivate the bacteria at 37°C and shock at 250 rpm/min overnight.

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