Alice:LabNotes/2010-4-9: Difference between revisions
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>Zsakura2 (→PCR) |
>Zsakura2 (→PCR) |
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*The DNA size is 200-500bp, so we use 3-10ng of DNA in 8ul volume | *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 1ul of purified PCR product with 7ul of ddH2O. | ||
*Add the Forward primer for sequencing, dilute the Forward primer to | *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 15ul. |
Revision as of 17:30, 12 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 94ul 2ng/ul
PCR
- reactions total.
DNA(2ng/ul) 3ul Taq 2x master mix 25ul 10uM primer 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 5uM, add 5ul of primer(total of 25pmol) and get a final volume of 15ul.