Kun:LabNotes/ASE/2009-7-20: Difference between revisions

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(New page: ==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 ...)
 
Line 12: Line 12:
   H2O          9ul          11ul
   H2O          9ul          11ul


   95C 2min -> -0.02C/sec to 58C -> 58C 16h  
   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)  
       -> 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.
       -> 58C 2h -> 94C 1min -> 37C 1min -> add 2ul Exo I & III (1ul RNase H to B) -> 37C 2h -> 4C hold.

Revision as of 23:10, 20 July 2009

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 AmpF6.2Sol       0.4ul    
  100uM AmpR6.2Sol       0.4ul    
  50X SYBG I:            0.4ul    
  H2O                     38ul    

98C 30sec -> (98C 10sec -> 54C 30sec -> 72C 15sec) x 8 -> (98C 10sec -> 72C 20sec) x 15 -> 72C 3min -> 4C hold.