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

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>Zsakura2
>Zsakura2
Line 11: Line 11:
     DNA(2ng/ul)              3ul   
     DNA(2ng/ul)              3ul   
     Taq 2x master mix      25ul
     Taq 2x master mix      25ul
     10uM primer (SPEN)      1ul   
     10uM primer           1ul   
     H2O                    21ul
     H2O                    21ul
     total                  50ul
     total                  50ul

Revision as of 16:25, 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 2uM, add 4ul of primer and get a final volume of 12ul.