Team:USTC/Notebook/6 16
Notebook
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Performers
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
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.
