Alice:LabNotes/2010-4-9: Difference between revisions

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==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.

Latest revision as of 17:30, 12 April 2010