Modeling

Everybody

Date: June 6th by Chengle Zhang

Last growth curve looks terrible， so we decide to repeat the experiment.

Yeast Growth Curve Measurement 2

Experimental materials:
YPD culture medium 500 mL(newly made by ourselves) Yeast W303(made by ourselves)

Procedure: 1. Pick the colonies of Yeast W303 into an EP tube containing some YPD culture,then shake it over till evenly. 2. Take 1 mL liquid from the EP tube to three 250 mL erlenmeyer flasks respectively,each containing 100 mL YPD culture already. 3. Cultivate them at 30°C and shake at 250 rpm/min,time from now. 4. Take 700 μL liquid from each of the three flasks into EP tube respectively after specific time(shown in the table below). 5. Measure the OD data of each EP tube.

Results:

Time point	Time/min	OD600 Sample1	OD600 Sample2	OD600 Sample3	OD600 Average
-8:45	0	0	0	0
	900	0.032	0.031	0.030	0.031
	1050	0.120	0.123	0.130	0.124
	1175	0.325	0.334	0.342	0.334
	1290	1.020	1.017	1.074	1.037
	1410	2.580	2.537	2.672	2.596
	1530	4.663	4.610	4.870	4.714
	1650	6.160	6.156	6.480	6.265
	2160	11.440	12.475	12.650	12.188
	2250	13.725	13.733	13.725	13.728
	2375	14.892	14.408	14.158	14.486
	2442	14.433	13.900	15.333	14.556
	2562	14.192	14.858	14.333	14.461
	2682	14.717	14.683	14.475	14.625

Use the data above to make a smooth curve:X-axis stands for time,Y-axis stands for OD600 Average. 6/6/2016 Yeast Growth Curve

PCR of R9

Date: June 9th Recorder：Tianshu Liu

Experiment Materials plasma backbone：PSB1C3 primer：r9-r r9-p 10uM sterilized ddH₂O Pfu 2xHIFI-PCR Master Mix

Procedure：

4 parallel trials

sample	volume
Pfu 2xHIFI-PCR Master Mix	10 μL
sterilized ddH₂O	7 μL
PSB1C3	1 μL
r9-r	1 μL
r9-p	1 μL
total	20 μL

PCR steps:

temperature	time
95	5 min
95	1 min
55	1 min
72	1 min
step 2- step 4	*25
72	10 min
4	24 h

Agarose gel electrophoresis gel: 1.1 g agarose per 100 mL 1XTAE buffer. 2.Dissolved by heating. 3.Cool down. 4.Pour the liquid into electrophoresis tank until solidification. 5.Mix 5 μL PCR product with 1μL 6Xloading buffer. Loading：Trans2K Plus II DNA marker 5μL, sample 6 μL. 6.Electrophoresis gel：110 V for 30 min 7.Dissolve 10000XGelred to a container and put the agarose gel in. 8.Autoradiography(UV).

Result Failed，marker is the only thing which is visable.

Possible Reasons

1.DNA template has denaturated. 2.Primer mismatch. 3.DNA template is sufficient . 4.Primer TM is too high to separate. 5.PCR cycling conditions are not suitable. 6.Temperature is too high to connect primer to plasma backbone.

Date: June 9th

PCR of sup35

Recorder: Xuefeng Meng

Experimental materials Saccharomyces cerevisiae genome(borrowed from Miss Wu), primer 1 & primer 2(10 μM), Sterilized ddH₂O, 2×HiFi-PCR Master About primers Designed by ourselves.

primer	1	2
sequence	5'-ATGTCGGATTCAAACCAGGGCAAC-3'	5'-CATTCACCACCTCATCGTCAACTTCC-3'
lenth (nt)	24	26
GC%	50	50
molecular weight (Da)	7370.9	7746.1
Tm (℃)	61	61

Procedure:

1.Prepare a PCR tube and sequentially add：

sample	volume
Sterilized ddH₂O	7 μL
2×HiFi-PCR Master	10 μL
SC genome	1 μL
Primer 1	1 μL
Primer 2	1 μL
total	20 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	45 s
step 3	55	1 min
step 4	72	1 min
step 5	72	7 min
step 6	4	--

30 cycles(step 2 ~ step 4) four parallel group trial

3.Agarose gel electrophoresis gel: 3.1.Add 1 g agarose to 100 mL TAE buffer. 3.2.Dissolved by heating. 3.3.Cool down. 3.4.Pour into electrophoresis tank. 3.5.Mix 5 μL PCR product and 1 μL loading buffer. 3.6.Loading：Trans2K Plus II DNA marker 5 μL，sample 6 μL. 3.6.Electrophoresis gel：110 V 30 min. 3.7.Dissolve Gelred to a container and put the agarose gel in. 3.8.Autoradiography(UV).

Result Failed only marker is visable

Possible reasons

DNA template has denaturated.
The amount of DNA template is too little.
Primer is not suitable.
PCR cycling conditions(temperature and time) are not suitable.
Operational errors.

We need more Repeated experiments.

Date: June 12th

Recorder: Yonghao Liang

Transformation of J04450

experimental materials

competent E·coil BL21 100 μL（bought from Transgen Biotech)

J04450-pSB1C3 plasmid 2 μL（50 pg/μL）

procedure:

Spin down the DNA tubes from the Competent Cell Test Kit/Transformation Efficiency Kit to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient. Note: There should be 50 µL of DNA in each tube sent in the Kit.
Thaw competent cells on ice. Then pre-chill by placing the tubes on ice.
Pipet 2 µL of DNA into competent cell tube.
Pipet 100 µL of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!
Heat-shock the cells by placing into the waterbath for 1 minute. 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 900 µL of LB media per tube, and incubate at 37°C for 2 hours.
Centrifuge the tube at 4000 rpm for 2 minutes，make the bacteria deposit at the bottom of the tube.
Discard 800 μL supernatant liquid and resuspend the bacteria .

2. Coat plate: add the solution 200 μL in a plate and spread it , then cultivate it overnight.

Date: June 13th

Recorder:Ya Jiang

Colony picking

Add 5mL LB liquid medium (1.7 μL 100 mg/mL chloramphenicol has already been in LB) in each tube and we prepare 4 tubes.
Pick the colnes in the test tubes.

Date: June 13

PCR of sup35

Recorder: Xuefeng Meng

Experimental materials Saccharomyces cerevisiae genome(borrowed from Miss Wu), primer 1 & primer 2(10 μM), Sterilized ddH₂O, 2×HiFi-PCR Master，MgCl₂(5 μM)

Procedure:

1.Prepare 4 PCR tubes and sequentially add：

sample	volume1	volume2	volume3	volume4
Sterilized ddH₂O	6 μL	6 μL	6 μL	5.5 μL
2×HiFi-PCR Master	10 μL	10 μL	10 μL	10 μL
MgCl₂	1 uL	0	0	0
SC genome	1 μL	2 μL	1 μL	1.5 μL
Primer 1	1 μL	1 μL	1.5 μL	1.5 μL
Primer 2	1 μL	1 μL	1.5 μL	1.5 μL
total	20 μL	20 μL	20 μL	20 μL

2.PCR reaction Parameters setting：

stage	temperature	time
step 1	95	5 min
step 2	95	1 min
step 3	55	1 min 50 s
step 4	72	1 min 50 s
step 5	72	10 min
step 6	4	--

30 cycles(step 2 ~ step 4)

Date: June 14

PCR of sup35

Recorder: Xuefeng Meng

Experimental materials agarose TAE buffer loading buffer gelred Trans2K Plus II DNA marker PCR products

Procedure: Agarose gel electrophoresis gel:

Add 0.5 g agarose to 50 mL TAE buffer.
Dissolved by heating.
Cool down.
Pour into electrophoresis tank.
Add 1 μL Gelred into 999 μL loading buffer.
Mix 5 μL PCR product and 1 μL loading buffer(containing Gelred).
Mix 5 μL DNA marker and 1 μL loading buffer(containing Gelred).
Loading：DNA marker 6 μL, sample(*4) 6 μL.
Electrophoresis gel：110 V 30 min.
Autoradiography(UV).

Result Failed only marker is visable.

3. Cultivate the bacteria at 37 degree Celsius centigrade and shock at 250 rpm/min overnight.

Date: June 14th

Recorder: Ya Jiang

Plasmid Extraction for J04450

1 Centifuge 1.5 mL bacterium solution at 11000 rpm, few sediment getted. Remove the supernatant. Add another 1.5 mL bacterium solution to the EP tube and centifuge at 11000 rpm again.

2 Add 250 μL Buffer P1, resuspend cells.

3 Add 250 μL Buffer P2, mix well, 4 min's standing.

4 Add 350 μL Buffer P3, mix well.

5 134000 rpm centifuge 10 min, move all supernatant to adsorption column, 11.6 rpm centifuge 30 s, discard filtrate.

6 Add 500 μL Buffer DW1, 12000 rpm centifuge 30 s, discard filtrate.

7 Add 500 μL Wash Solution, 12000 rpm centifuge 30 s, discard filtrate. Repeat once.

8 12000 rpm centifuge 1 min.

9 Put the adsorption column in a new EP tube. Add 50 μl ddH2O( heating in water bath at 55 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 measure the OD and A260/A280 of plasmids. The results demonstrate that plasmid extraction is successful.