Team:USTC/Notebook/8 9

Modeling

Everybody

Date: 8.9

Recorder: Kaiyue Ma Ligation of pro+RBS+GFP1-10 & pSB1A3 The concentration of the insert DNA is 60.4 ng/μL. The concentration of the vector DNA is less than 34.6 ng/μL.

material	volume
Linear vector DNA	10 μL
Insert DNA	7.8 μL
10X buffer	2 μL
T4 DNA ligase	0.2 μL
Total	20 μL

Incubate 30 min at 22 °C.

Plasmid Extraction for PSB1A3 Recorder: Kaiyue Ma & Junjie Zeng & Yonghao Liang

sample	1	2	3	4	5	6	7
A260/A280	1.90	1.89	1.80	1.96	1.87	1.86	1.90
A260/A230	1.84	1.98	1.40	0.9	2.15	1.79	2.33
concentration(ng/μL)	119.0	149.5	247.6	20.2	436.9	146.9	180.9

Recorder: Kaiyue Ma Double digestion of pSB1A3 Materials:

Material	volume
dd water	14.35 μL
10X buffer	2 μL
plasmid (436.9 ng/μL)	2.3 μL
FD EcoR I	1 μL
FD Pst I	0.35 μL
pure love	a lot
total	20 μL

Agarose gel electrophoresis Result: 图片名称 (PS: 0 is the control group(5 μL plasmid + 1 μL loading buffer), 1 to 2 are the digest products(20 μL products + 4 μL loading buffer), 3 is the marker) We did gel extration.

The result are as follows:

sample	1
A260/A280	1.62
A260/A230	0.12
concentration(ng/μL)	20.6

Recorder: Yonghao Liang **Transformation of recombinant plasmid of pSB1A3+PRORBS+sfGFP1-10 **

Date: 8.9 Plasmid Extraction for PSB1A3 Recorder:Chenyang Li and Weijie Jin

Special notes:using visualize material.

sample	1	2	3	4	5
A260/A280	1.86	1.88	1.83	1.86	1.86
A260/A230	1.38	2.21	1.76	1.94	1.95
concentration(ng/μL)	7.2	274.1	329.4	328.3	225.6

(note: Sample 1 may not be used because its concentration is too low.)

Recorder: Kaiyue Ma Colony picking of pYeWGAP-GFP1-10 & pYeWGAP-AD Same as usual

Recorder: Chenyang Li, Yonghao Liang Ligation of sfGFP1-10 and PSB1A3

Material: 1. double digestion product of sfGFP1-10 2. double digestion product of PSB1A3 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: Add to the sample1: 5 μL sfGFP1-10 10μL PSB1A3 2.8 μL nuclease-free water 2 μL T4 DNA Ligase Buffer 0.2 μL T4 DNA Ligase Add to the sample2: 2.5 μL sfGFP1-10 8 μL PSB1A3 7.3 μL nuclease-free water 2 μL T4 DNA Ligase Buffer 0.2 μL T4 DNA Ligase

Mix gently and incubate at 22 degree Celsius for 0.5 hour.

Recorder: Yonghao Liang **Transformation of recombinant plasmid of pSB1A3+PRORBS+sfGFP1-10 **

Recorder: Junjie Zeng
PCR of sfGFP11

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1-4
Sterilized ddH₂O	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Template (sfGFP)	1 μL
standard-biobrick-p	1 μL
standard-biobrick-r	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction

Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	12 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Date: 8.10

Recorder: Junjie Zeng
**Gel Extraction of PCR product **[

Recorder: Chenyang Li PCR of pYeWGAP-pGAL-GFP, pYeWGAP-AD and pYeWGAP-BD

Procedure:

1.Prepare 2 PCR of pYeWGAP-pGAL-GFP tubes and sequentially add： 10 μL Sterilized ddH₂O, 12.5 μL Taq PCR Master Mix, 1 μL primer F, 1 μL primer R, 0.5 μL DNA template.

2.Prepare 2 PCR of pYeWGAP-AD tubes and sequentially add： 9 μL Sterilized ddH₂O, 12.5 μL Taq PCR Master Mix, 1 μL primer F, 1 μL primer R, 1.5 μL DNA template.

3.Prepare 2 PCR of pYeWGAP-BD tubes and sequentially add： 9 μL Sterilized ddH₂O, 12.5 μL Taq PCR Master Mix, 1 μL primer F, 1 μL primer R, 1.5 μL DNA template.

4.PCR reaction Parameters setting：

stage	temperature	time
step 1	94	10 min
step 2	94	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)

Recorder：Chenyang Li Double Digestion of GFP

Procedure: In either of the samples of GFP, add nuclease-free water 11.65 μL 10*Green Buffer 2 μL plasmid 5 μL EcoRI 1 μL PstI 0.35 μL Mix gently and incubate at 37 degree Celsius for 15 min.

result of Digestion of GFP and PCR of AD, BD and PYW from left to right: Trans 2K plus(contain Gelred), GFP from gel extraction, trans 2K plus, GFP from digestion, trans 2K(contain Gelred), AD1, AD3, AD4, PYW1, PYW2, AD5 and BD5

Recorder：Yonghao Liang Colony picking of combinant plasmid of PRORBS+PR(EcPR)

Recorder: Yonghao Liang Transformation of plasmid pET28a + SUP35

Recorder: Kaiyue Ma Purification of PCR product of sfGFP11 Using the kit bought from Sangon Biotech, the procedure is same as before. Code four tubes as 0101, 0102, 0103 and 0104.

Recorder: Yonghao Liang PCR of SUP35

Procedure:

1.Prepare 4 PCR tubes and sequentially add： 32.5 μL Sterilized ddH₂O, 25 μL 5X PS Buffer(the volumn here is wrong), 4 μL dNTP, 0.5 μL PrimeStar, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.About primers

primer	1	2
sequence	5'-CCGGAATTCGCGGCCGCTTCTAGATGGGCGATTCAAACCAAGGCAACA-3'	5'-TGCACTGCAGCGGCCGCTACTAGTAATCGTTAACAACTTCGTCATCCACTTCT-3'

3.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	7 min
step 2	95	10 s
step 3	57	15 s
step 4	72	50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

4.Agarose gel electrophoresis Result: 图片名称

5.Then we did gel extration.The result is as follows:

sample	1	2	3	4
A260/A280	1.86	1.84	1.77	1.82
A260/A230	0.76	0.36	0.31	0.25
concentration(ng/μL)	137.3	120.7	60.7	25.6

Recorder：Chenyang Li Double Digestion of AD-PYT-4 and BD-PYT-1 1.Double Digestion of AD-PYT-4 Procedure: In either of the samples of AD-PYT-4, add 10Green Buffer 2 μL plasmid 17 μL EcoRI 0.25 μL PstI 0.75 μL Mix gently and incubate at 37 degree Celsius for 45 min. It's failed because the volume of EcoRI and PstI are confused. 2.Double Digestion of BD-PYT-1 Procedure: In either of the samples of BD-PYT-1, add nuclease-free water 2 μL 10Green Buffer 1 μL plasmid 6 μL EcoRI 0.25 μL PstI 0.75 μL Mix gently and incubate at 37 degree Celsius for 45 min. It's failed because the volume of EcoRI and PstI are confused. There are another two correct procedure. 3.Double Digestion of AD-PYT-4 Procedure: In either of the samples of AD-PYT-4, add 10Green Buffer 2 μL plasmid 17 μL EcoRI 0.75 μL PstI 0.25 μL Mix gently and incubate at 37 degree Celsius for 15 min. 4.Double Digestion of BD-PYT-1 Procedure: In either of the samples of BD-PYT-1, add nuclease-free water 2 μL 10Green Buffer 1 μL plasmid 6 μL EcoRI 0.75 μL PstI 0.25 μL Mix gently and incubate at 37 degree Celsius for 15 min. result of Digestion of all the above. Plasmid Extraction for AD-RYT-4 and BD-PYT-1 from left to right: trans 2K(contain Gelred), product of the second double digestion of BD-PYT-1,product of the second double digestion of BD-PYT-1,product of the first double digestion of AD-PYT-1 and product of the first double digestion of AD-PYT-1

Plasmid Extraction for AD-RYT-4 and BD-PYT-1 Recorder: Yinchenguang Lyu

number	AD-PYT-4	BD-PYT-1
A260/A280	1.74	1.64
A260/A230	1.14	1.79
concentration(ng/μL)	45.6	166.3

Recorder: Yonghao Liang PCR of SUP35

Procedure:

1.Prepare 4 PCR tubes and sequentially add： 32.5 μL Sterilized ddH₂O, 10 μL 5X PS Buffer, 4 μL dNTP, 0.5 μL PrimeStar, 1 μL template, 1 μL primer 1, 1 μL primer 2.

2.About primers

primer	1	2
sequence	5'-CCGGAATTCGCGGCCGCTTCTAGATGGGCGATTCAAACCAAGGCAACA-3'	5'-TGCACTGCAGCGGCCGCTACTAGTAATCGTTAACAACTTCGTCATCCACTTCT-3'

3.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	7 min
step 2	95	10 s
step 3	57	15 s
step 4	72	50 s
step 5	72	10 min
step 6	4	--

35 cycles(step 2 ~ step 4)

4.Agarose gel electrophoresis Result: 图片名称

5.Then we did gel extration.The result is as follows:

sample	1	2	3	4
A260/A280	1.70	1.86	1.85	1.85
A260/A230	0.30	0.39	0.41	1.74
concentration(ng/μL)	59.1	156.5	143.2	194.5

Recorder：Chenyang Li Ligation of PYescGAP and pGal1-GFP,PYescGAP and AD,PYescGAP and BD

Reaction system: 1.5 μL PYescGAP, 2 μL 10× T4 DNA ligase buffer, 0.2 μL T4 DNA ligase, 3.2 μL pGal1-GFP(29.1 ng/μL)/1 μL AD(30.0 ng/μL)/1 μL BD(31.0 ng/μL), 13.1 μL/15.3 μL/15.3 μL nuclease-free water

Mix gently and incubate at 22 degree Celsius for 20 mins.

Then we do transformation of combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD, PYescGAP and BD.

Recorder: Yu Xie PCR of bacteria containing recombinant plasmid pYeT containing PRsfGFP1-10

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1,2,3,4
Sterilized ddH₂O	9 μL
Taq PCR Master Mix	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	94	4 min
Duration	94	30 s
Anneal	55	30 s
Extend	72	42 s
Post-Extend	72	10 min
Final	4	--

35 cycles(Duration ~ Extend)

Recorder: Ya Jiang & Yu Xie PCR of sfGFP1-10

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	1-4
Sterilized ddH₂O	32.5 μL
5×PrimeSTAR Buffer (Mg²⁺Plus)	10 μL
dNTP Mixture (2.5 mM each)	4 μL
Template (sfGFP)	1 μL
standard-sfGFP1-10-p	1 μL
standard-sfGFP1-10-r	1 μL
PrimeSTAR HS DNA Polymerase (2.5 U/μl)	0.5 μL
total	50 μL

2.PCR reaction

Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	10 s
step 3	55	15 s
step 4	72	43 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

We did agarose gel electrophoresis for check The results are as follows: 图片名称 which shows our PCR results are successful.

Then we did purification of PCR product of sfGFP1-10 We have 50 μL solution in each tube to be purified by PCR purification kit (bought from Sangon Biotech). And the procedure is showed below:

Add 250 μL Buffer B3 to the 50 μL solution and mix it up. Add it to an adsorption column.
11,000 rpm centrifuge for 30 s, and then discard the filtrate.
Add 500 μL Wash Solution, 12,000 rpm centrifuge for 30 s, and then discard the filtrate.
Repeat last process.
Centrifuge the empty column at 12,000 rpm for 1 min.
Lie the column still for 10 min.
Put the column to an 1.5 ml EP tube, add 25 μL ddH₂O(55°C), stand them still for 10 min. Centrifuge at 12,000 rpm for 1 min.

Results:

sample	sfGFP1-10-1	sfGFP1-10-2	sfGFP1-10-3	sfGFP1-10-4
A260/A280	1.90	1.91	1.75	1.63
Concentration(ng/μl)	140.0	143.0	33.3	34.5

Recorder: Yu Xie Double digestion of pYeT and sfGFP1-10 Reaction system:

Sample	11μL sfGFP1-10-1	11μL sfGFP1-10-2	11μL sfGFP1-10-3	11μL sfGFP1-10-4	15μL pYeT-1	15μL pYeT-2
nuclease-free water(μL)	4	4	4	4	9	9
fastdigest buffer(μL)	2	2	2	2	3	3
PstI(μL)	1	1	1	1	1.5	1.5
EcoRI(μL)	1	1	1	1	1.5	1.5
total(μL)	20	20	20	20	30	30

Mix gently and incubate at 37 degree Celsius for 30 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1,,sample2,sample3,sample4,control-1,sample5,sample6,control-2)

We did gel extration.The result are as follows:

sample	sfGFP1-10-1(cut)	pYeT
A260/A230	1.51	1.11
A260/A280	1.92	1.85
concentration(ng/μL)	65.5	558.8

Recorder: Yu Xie Ligation of pYeT and sfGFP1-10

Material: 1. double digestion product of PYescGAP 2. double digestion product of sfGFP1-10 3. 10× T4 DNA ligase buffer,T4 DNA ligase(bought from Thermo Fisher Scientific) 4. nuclease-free water

Procedure: 1.Add to either of samples:

Sample	volume
PYescGAP (cut)	0.3μL
sfGFP1-10	1μL
nuclease free water	16.4μL
10* T4 DNA Ligase Buffer	2.0μL
T4 DNA Ligase	0.3μL
total	20μL

2.Mix gently and incubate at 22 degree Celsius for 0.5 hour.

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

Recorder: Yonghao Liang & Yinchenguang Lyu Double digestion of sup35 Reaction system: 1.2 μL PCR products, 25.1 μL nuclease-free water, 2 μL fastdigest greenbuffer, 1.35 μL EcoRI, 0.35 μL Bcu1.

Mix gently and incubate at 37 degree Celsius for 15 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1,,sample2,sample3,sample4)

We did gel extration.The result are as follows:

sample	1	2
concentration(ng/μL)	8.0	3.0

DATE 8.11

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.

Recorder：Chenyang Li and chengle Zhang Ligation of PYescGAP and pGal1-GFP,PYescGAP and AD,PYescGAP and BD

Reaction system: 1.5 μL PYescGAP, 2 μL 10× T4 DNA ligase buffer, 0.2 μL T4 DNA ligase, 3.2 μL pGal1-GFP(29.1 ng/μL)/1 μL AD(30.0 ng/μL)/1 μL BD(31.0 ng/μL), 13.1 μL/15.3 μL/15.3 μL nuclease-free water

Mix gently and incubate at 22 degree Celsius for 20 min.

Agarose gel electrophoresis Result:

Then we do transformation of combinant plasmid of PYescGAP and pGal1-GFP, PYescGAP and AD, PYescGAP and BD.

results

sample	1	2
A260/A280	1.85	1.88
A260/A230	0.58	0.18
concentration(ng/μL)	234.9	67.8

Recorder: Yonghao Liang & Yinchenguang Lyu Double digestion of sup35 Reaction system: 1.2 μL PCR products, 25.1 μL nuclease-free water, 2 μL fastdigest greenbuffer, 1.35 μL EcoRI, 0.35 μL Bcu1.

Mix gently and incubate at 37 degree Celsius for 15 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(Transplus 2K),sample1(hole5),sample2(hole6),sample3(hole7),sample4(hole8))

We did gel extration.The result are as follows:

sample	1	2
A260/A280	2.01	1.85
A260/A230	0.10	0.06
concentration(ng/μL)	8.4	9.3

(sample 2 comes from the second time we elute the tube)

Recorder: Yonghao Liang & Yinchenguang Lyu Double digestion of sup35 Reaction system: 20 μL PCR products, 5.25 μL nuclease-free water, 3 μL fastdigest greenbuffer, 1.3 μL EcoRI, 0.45 μL Bcu1.

Mix gently and incubate at 37 degree Celsius for 15 mins .

Agarose gel electrophoresis Result: 图片名称 (from left to right:marker(2K),marker(2k plus 2),sample1,,sample2,control group)

We did gel extration.The result are as follows:

sample	1	2
A260/A280	29.9	1.85
A260/A230	0.08	0.39
concentration(ng/μL)	29.9	30.1

(sample 2 comes from the second time we elute the tube)

Recorder: 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	16.25 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	25.5 μL