Team:USTC/Notebook/9 1

Modeling

Everybody

Date: 9.1

Double Digestion of YeWGAP

Recorder: Yinchenguang Lyu

3 μL template 1 μL EcoRI 0.35 μL Pstl 2 μL 10×FastDigest Buffer 13.65 μL ddH₂O

result

sample	YeWGAP(digested)
A260/A280	1.81
A260/A230	0.91
concentration(ng/μL)	72.5

Recorder：Tianshu Liu

Double Digestion of sfGFP1-10

Materials:

1. sfGFP1-10-PSB1C3 plasmid extract from E.coli

2. Restriction enzyme (Fastdigest) EcoRI, PsI and 10×Green Buffer(bought from Thermo Fisher Scientific)

3. Nuclease-free water

Procedure:

Add the materials into new EP tubes in the order of following table respectively.

sample	10 μL sfGFP1-10-PSB1C3
10×Green Buffer(μL)	2
nuclease-free water(μL)	6.65
EcoRI(μL)	1
PstI(μL)	0.35
total(μL)	20

Mix gently and incubate at 37 degree Celsius for 30 mins

Recorder：Tianshu Liu

Agarose gel electrophoresis of digestion sfGFP1-10

Materials:

1. 1×TAE buffer

2. DNA marker （Trans 2K Plus II）

3. 6× loading buffer

Procedure:

1. Add 0.5 g agarose to 50 mL 1*TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Add 4 μL 6× loading buffer to 20 μL digestion product, add 2 μL 6× loading buffer to 2 μL GFP plasmid with 8μL water as control group. 6. Loading： Plate: DNA marker 5 μL（Trans 2K Plus II), sample on the left are 20 μL digestion product, sample on the right is control group. 7. Electrophoresis gel：110 V 30 min. 8. Autoradiography(UV).

Recorder: Tianshu Liu

Gel Extraction of sfGFP1-10-PSB1C3 double digestion products

Procedure: 1. Cut off the Gel part containing the target band. Get 2 tubes of pTEF2 double digestion products. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of pGal1&GFP.

Result

sample	sfGFP1-10
concentration(ng/μL)	24.4

Recorder：Tianshu Liu Double Digestion of PSB1A3 Materials: 1. sfGFP1-10-PSB1C3 plasmid extract from E.coli 2. Restriction enzyme (Fastdigest) EcoRI, PsI and 10×Green Buffer(bought from Thermo Fisher Scientific) 3. Nuclease-free water

Procedure: Add the materials into new EP tubes in the order of following table respectively.

sample	7 μL PSB1A3
10×Green Buffer(μL)	2
nuclease-free water(μL)	9.65
EcoRI(μL)	1
PstI(μL)	0.35
total(μL)	20

Mix gently and incubate at 37 degree Celsius for 30 mins

Recorder：Tianshu Liu Agarose gel electrophoresis of digestion PSB1A3 Materials: 1. 1×TAE buffer 2. DNA marker （Trans 2K Plus II） 3. 6× loading buffer

Procedure: 1. Add 0.5 g agarose to 50 mL 1*TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Add 4 μL 6× loading buffer to 20 μL digestion product, add 2 μL 6× loading buffer to 2 μL GFP plasmid with 8μL water as control group. 6. Loading： Plate: DNA marker 5 μL（Trans 2K Plus II), sample on the left are 20 μL digestion product, sample on the right is control group. 7. Electrophoresis gel：110 V 30 min. 8. Autoradiography(UV).

Recorder: Tianshu Liu Gel Extraction of PSB1A3 double digestion products

Procedure: 1. Cut off the Gel part containing the target band. Get 2 tubes of pTEF2 double digestion products. 2. Weigh the cut off Gel part. 3. Add Buffer B2 whose weight is 3 times of the Gel. 4. Add isopropanol whose volume is 1/3 of the Buffer B2. 5. Move the solution to adsorption column , 11000 rpm centifuge 30 s. 6.Put filtrate into the upper part of the same adsorption column,11000 rpm centifuge 30,discard filtrate. 7. Add 300 μL Buffer B2,11000 rpm centifuge 30 s, discard filtrate. 8. Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once. 9. 12000 rpm centifuge 1 min. 10. Lying for 10 min. 11. Put the adsorption column in a new EP tube. Add 20 μL elution buffer which is pre-heated at 60 degree Celsius , 10 min's standing, 12000 rpm centifuge 1 min. Preserve in the EP tube with ddH₂O at 4 degree Celsius.

After extraction, we measured the OD A260/A280 and the concentration of pGal1&GFP.

Result

sample	PSB1A3
concentration(ng/μL)	16.0

Date:9.2

Recorder: Tianshu Liu Transformation of recombinant plasmid of sfGFP1-10 & PSB1A3 Materials: recombinant plasmid of sfGFP1-10 & PSB1A3 LB media competent cells (E.coli Top 10)

Procedure:

Put competent cells on ice. Then pre-chill by placing the tubes on ice. Pipet the recombinant plasmid into competent cell tubes, 10 µL for each. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. Heat-shock the cells by placing into the waterbath for 90 seconds. Be careful to keep the lids of the tubes above the water level, and keep the ice close by. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover. Add 1000 µL of LB media per tube, and incubate at 37°C for 1 hours. Centrifuge the tube at 4000 rpm for 1 minutes, make the bacteria deposit at the bottom of the tube. Discard 800 μL supernatant liquid and resuspend the bacteria. Coat plate: add the solution to plate (Ap+) and spread it, respectively, then cultivate it overnight.

Date: 9.5

Recorder: Kaiyue Ma

We examined the sequence of the SUP35NM-sfGFP11 ligation, and the results show that we succeeded. Now we should do the site mutation to make it work.

Date: 9.6

Recorder: Kaiyue Ma

The site mutations of the SUP35NM-sfGFP11 ligation succeeded. We did one of the site mutations to eliminate the stop codon in the middle, and did the other to eliminate the PstI site in the middle.

Recorder: Chenyang Li, Yinchenguang Lyu , Xuefeng Meng,Ya Jiang ,Chengle Zhang etc. During the date 8.21-9.6,we accomplished the following experiments: 1. Ligation of AD/BD and SUP35NM in pSB1C3 plasmid; 2. Site mutations of the SUP35NM-AD/BD ligation . (One of the site mutations is aimed to eliminate the stop codon in the middle, and the other to eliminate the PstI site in the middle.) 3. Double digestion of combinant pSB1C3 plasmid of AD/BD and SUP35NM with EcoRI and PstI. Ligation of the digestion product and plasmid PYeScGAP that is digested with the same enzyme. 4. Ligation of pGal1-GFP and plasmid PYeLGAP/pSB1C3 that are both digested with EcoRI and PstI. 5. Site mutation of the ligation product in 4 which is aimed at eliminating the stop codon in the middle.

Date: 9.7

Recorder: Ya Jiang

To verify the split GFP can function as our expectation, we transform the plamids containing sfGFP1-10 and the plamids containing sfGFP11 respectively in BL21, cultivating the bacteria at 37°C and shacking at 250 rpm/min overnight. We use fluorescence microscope to observe bacterias under 100X objective lens.