Alice:LabNotes/2009-12-3

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Sanger validation of candidate mutations[edit]

DNA preparation[edit]

  • prepared the following stock solutions for PCR templates
    Sample     Conc.       Volume      H2O       Final Conc.
    CV-iPS F   309ng/ul      1ul      153.5ul    2ng/ul
    CV-ips B   358ng/ul      1ul      178ul      2ng/ul  
    CV-Fibr    712ng/ul      1ul      355ul      2ng/ul

PCR[edit]

  • Primers and templates:
' CV-fibroblast CV-ips-B CV-ips-F
MCM3 x x x
18-22778270 x
ZSCAN x x x
C1orf x x x
SPEN x x x
  • 13 reactions total.
   DNA(2ng/ul)              3ul  
   Taq 2x master mix       25ul
   10uM Primer mix          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:
File:ZhangLab 2 2009-12-04 10hr 21min.jpg  File:ZhangLab 2 2009-12-03 15hr 14min.jpg
  • 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
' CV-fibroblast (DNA+H2O) CV-ips-B (DNA+H2O) CV-ips-F (DNA+H2O)
MCM3 1ul + 7ul 2ul + 6ul 1ul + 7ul
18-22778270 1ul + 7ul
ZSCAN 1.2ul + 6.8ul 1.5ul + 6.5ul 1.5ul + 6.5ul
C1orf 1ul + 7ul 1.1ul + 6.9ul 1.2ul + 6.8ul
SPEN 2ul + 6ul 1.1ul + 6.9ul 2ul + 6ul
  • Add the Forward primer for sequencing, dilute the Forward primer to 2uM, add 4ul of primer and get a final volume of 12ul.


  • [[Media:|Raw ABI trace file]].