Date: June 16

PCR of sup35

Recorder: Xuefeng Meng；Junjie Zeng; Jianyi Wang

Experimental materials Saccharomyces cerevisiae genome(10 times dilution, 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	volume6	volume6
Sterilized ddH2O	2 μL	4 μL	5 μL	5 μL	4 μL	5 μL
2×HiFi-PCR Master	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL
MgCl2	1 μL	0	0	1 μL	1 μL	0
SC genome	5 μL	4 μL	3 μL	2 μL	3 μL	2 μL
Primer 1	1 μL	1 μL	1 μL	1 μL	1 μL	1.5 μL
Primer 2	1 μL	1 μL	1 μL	1 μL	1 μL	1.5 μL
total	20 μL	20 μL	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	1.5 min
step 4	72	1.5 min
step 5	72	7 min
step 6	4	--

30 cycles(step 2 ~ step 4) Stored at 4°C.

Date: June 24

PCR of sup35

Recorder: Xuefeng Meng

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

Procedure: Agarose gel electrophoresis gel: 1. Add 1 g agarose to 100 mL TAE buffer. 2. Dissolved by heating. 3. Cool down. 4. Pour into electrophoresis tank. 5. Mix 5 μL PCR product and 1 μL loading buffer. 6. Loading：DNA marker 5 μL, sample(*6) 6 μL. 7. Electrophoresis gel：110 V 30 min. 8. Dissolve about 3 μL Gelred to a container with TAE buffer and put the agarose gel in. 10. Autoradiography(UV).

Result Failed

Advices 1. Prepare the agarose gel in advance and store it at 4°C before using in case that the temperature is too high when conducting electrophoresis gel. 2. We'd better soak the agarose gel in the TAE with Gelred so that to get a better staining effect.