Kun:LabNotes/ASE/2009-7-20

From ZhangLabWiki
Revision as of 06:57, 21 July 2009 by KunZhang (talk | contribs) (→‎PCR)
Jump to navigation Jump to search

Test the idea of doing SNP capturing directly on RNA

  • Probes: CES8k18bp: 14ng/ul
  • Samples:
    • Hues6-ES ds-cDNA, 50ng/ul (2009-03-11)
    • Hues6-ES total RNA 781ng/ul
                A            B
             ds-cDNA     total RNA
             50ng/ul      781ng/ul
  Sample       6ul           4ul
  CES8k18bp    2ul           2ul
  10x buffer   2ul           2ul
  H2O          9ul          11ul
  94C 2min -> 58C 16h 
     -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul to A or MonsterScript RT 2U/ul to B, , AmpLigase 1U/ul) 
     -> 58C 2h -> 94C 1min -> 37C 1min -> add 2ul Exo I & III (1ul RNase H to B) -> 37C 2h -> 4C hold.

PCR

  PCR                           x 2 
  Template:               10ul     
  2X iProof MasterMix     50ul   
  100uM AmpF6.3rSol      0.4ul    
  100uM AmpR6.3rSol      0.4ul    
  50X SYBG I:            0.4ul    
  H2O                     38ul    

98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 15sec) x 8 -> (98C 10sec -> 72C 20sec) x 15 -> 72C 3min -> 4C hold.