Alice:LabNotes/2010-4-23: Difference between revisions
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=PCR re-amplification of the sequencing libraries with PAIR-END primer= | =PCR re-amplification of the sequencing libraries with PAIR-END primer= | ||
*samples to be re-amplified: | *samples to be re-amplified: | ||
*CViB: 5.27nM =10.43ng/ul | **CViB: 5.27nM =10.43ng/ul | ||
*DF: 81.7nM = 161.77ng/ul | **DF: 81.7nM = 161.77ng/ul | ||
*foreskin: 75.38nM = 149.25ng/ul | **foreskin: 75.38nM = 149.25ng/ul | ||
*ips17A: 2.04nM = 4.04ng/ul | **ips17A: 2.04nM = 4.04ng/ul | ||
*ips17B: 2.01nM = 3.9ng/ul | **ips17B: 2.01nM = 3.9ng/ul | ||
*ips11B: 3.81nM = 7.54ng/ul | **ips11B: 3.81nM = 7.54ng/ul | ||
*Hfib 11: 3.22nM = 6.38ng/ul | **Hfib 11: 3.22nM = 6.38ng/ul | ||
x4 well each | x4 well each |
Revision as of 23:47, 23 April 2010
PCR re-amplification of the sequencing libraries with PAIR-END primer
- samples to be re-amplified:
- CViB: 5.27nM =10.43ng/ul
- DF: 81.7nM = 161.77ng/ul
- foreskin: 75.38nM = 149.25ng/ul
- ips17A: 2.04nM = 4.04ng/ul
- ips17B: 2.01nM = 3.9ng/ul
- ips11B: 3.81nM = 7.54ng/ul
- Hfib 11: 3.22nM = 6.38ng/ul
x4 well each DNA Phusion HF PCR Master Mix (2x): 50ul PCR_R (100uM): 0.2ul PCR_F (100uM): 0.2ul SYBR Green 50x: 0.4ul H2O: 48ul Total: 100ul 98C 30sec -> (98C 10sec -> 55C 45sec -> 72C 30sec) x2 cycles -> (98C 10 sec -> 72C 30sec) x 3cycles -> 72C 5min -> hold at 4C
Sanger validation of candidate mutations
DNA preparation
- prepared the following stock solutions for PCR templates
Sample Conc. Volume H2O Final Conc. PGP1F 41.8ng/ul 2ul 39.8ul 2ng/ul PGP1-ips 399ng/ul 1ul 198.5ul 2ng/ul
PCR
- 6 reactions total.
x3 x2 DNA(2ng/ul) 3ul 15ul Taq 2x master mix 25ul 125ul 10uM F primer 1ul - 10uM R primer 1ul - H2O 20ul 100ul total 50ul 250ul 94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min.
- use 2ul to run a TBE gel to check the PCR results, then save 18ul for backup, and use the rest to do Qiaquick column purification elute in 30ul EB.
- Use Nanodrop to check the concentration of the purified PCR product.
- TBE gel result:
- Prepare the samples for sequencing:
- USE Pre-Mixed Samples Preparation Guidelines from Genewiz to mix the DNA and custom primers.
- The DNA size is 200-500bp, so we use 10ng of DNA in 10ul volume (~1ng/ul)
- Add 1ul of purified PCR product with 9ul of ddH2O.
- Add the Forward primer for sequencing, dilute the Forward primer to 5uM, add 5ul of primer(total of 25pmol) and get a final volume of 15