Alice:LabNotes/2010-4-9: Difference between revisions
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>Zsakura2 (Created page with '==Sanger validation of candidate mutations== ===DNA preparation=== * prepared the following stock solutions for PCR templates Sample Conc. Volume H2O Fi…') |
>Zsakura2 |
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===DNA preparation=== | ===DNA preparation=== | ||
* prepared the following stock solutions for PCR templates | * prepared the following stock solutions for PCR templates | ||
Sample Conc. | Sample Conc. Volume H2O Final Conc. | ||
CViB 358ng/ul 1ul 178ul 2ng/ul | CViB 358ng/ul 1ul 178ul 2ng/ul | ||
CVF-feb 190ng/ul 1ul | CVF-feb 190ng/ul 1ul 95ul 2ng/ul | ||
===PCR=== | ===PCR=== |
Revision as of 16:39, 9 April 2010
Sanger validation of candidate mutations
DNA preparation
- prepared the following stock solutions for PCR templates
Sample Conc. Volume H2O Final Conc. CViB 358ng/ul 1ul 178ul 2ng/ul CVF-feb 190ng/ul 1ul 95ul 2ng/ul
PCR
- reactions total.
DNA(2ng/ul) 3ul Taq 2x master mix 25ul 10uM primer (SPEN) 1ul H2O 21ul total 50ul 94C 2min -> 35x (94C 30s -> 57C 30s -> 72C 30s) -> 72C 3min.
- use 3ul to run a TBE gel to check the PCR results, then save 22ul 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 3-10ng of DNA in 8ul volume
- Add 1ul of purified PCR product with 7ul of ddH2O.
- Add the Forward primer for sequencing, dilute the Forward primer to 2uM, add 4ul of primer and get a final volume of 12ul.