Kun:LabNotes/ExonomeSeq/2009-3-3

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Revision as of 21:25, 5 March 2009 by KunZhang (talk | contribs) (→‎PCR)
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Test whether shaking can improve the circularization

Padlock capture

  • Probe sets:
    • Jan09-1: 46,417 probes, 334nM
    • Jan09-2: 53,493 probes, 294nM
    • Jan09-3: 53,828 probes, 186nM
  • Template DNA: PGP1, sheared with Bioruptor, 50ng/ul; 200ug per reaction.
  • Probe:target ratio: 200:1
                       Shaking Control      
   Template(50ng/ul)     4ul    4ul   
   10X AmpLigase buffer  2ul    2ul   
   Jan09-1 (334nM)      3.1ul  3.1ul     
   Jan09-2 (294nM)      4.0ul  4.0ul 
   Jan09-3(unknown)     4.0ul  4.0ul
   H2O                  2.0ul  2.0ul
   95C 2min -> -0.02C/sec to 60C -> 60C 18h 
     -> add 1ul SLN mix (dNTP 5uM, Stoffel 2U/ul, AmpLigase 1U/ul) 
     -> 60C 24h (move one tube to ThermoMixer @60C, 500rpm) 
     -> 94C 1min -> 37c 1min -> add 2ul Exo I&III
     -> 37C 2h -> 90C 5min -> 4C hold

PCR

  PCR                           x 2 x 2
  Template:               10ul
  2x iProof master mix:   50ul    
  100uM AmpF6.2          0.4ul    
  100uM AmpR6.2          0.4ul    
  50X SYBG I:            0.4ul    
  H2O                     39ul    
  98C 30sec -> (98C 10sec -> 53C 30sec -> 72C 15sec) x 16 -> 72C 3min -> 4C hold.
  Purified with Qiaquick columns, eluted with 30ul EB.
  Yield:
  PGP1-shaked: 28ng/ul x 30ul
  PGP1-CTRL:   24ng/ul x 30ul

2nd round PCR with Solexa adaptor

  PCR                           x 2 
  Template:                1ul
  2x iProof master mix:   50ul    
  100uM AmpF6.2Sol       0.4ul    
  100uM AmpR6.2Sol       0.4ul    
  H2O                     49ul    
  98C 30sec -> (98C 10sec -> 56C 30sec -> 72C 15sec) x 6 -> 72C 3min -> 4C hold.
  Purified with Qiaquick columns, eluted with 30ul EB.
  Yield:
  PGP1-shaked-LONG: 
  PGP1-CTRL-LONG: