Alice:LabNotes/2010-4-9

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Revision as of 17:30, 12 April 2010 by >Zsakura2 (→‎PCR)
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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       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.