Kun:LabNotes/ExonomeSeq/2009-10-16: Difference between revisions

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       PCR
       PCR
       Template              5ul      5ul
       Template              5ul      5ul      5ul
       2x Kapa Mix           50ul    50ul
       2x iProof Mix         50ul    50ul      50ul
       Solexa_PCR_up(100uM) 0.4ul    0.4ul
       Solexa_PCR_up(100uM) 0.4ul    0.4ul    0.4ul
       AmpR6.3Sol(100uM)    0.4ul      -
       AmpR6.3Sol(100uM)    0.4ul      -        -  
       AmpF6.3rSol(100uM)    -      0.4ul
       AmpF6.3rSol(100uM)    -      0.4ul      -
       H2O                  44ul    44ul
      50X SYBGI            0.4ul    0.4ul    0.4ul
       95C 30sec -> (95C 3sec -> 60C 30sec) x 18 -> 60C 2min -> 15C hold.
       H2O                  44ul    44ul      44ul
       98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 15sec) x 10 -> 72C 2min -> 15C hold.


      
      
Line 40: Line 41:
       2x QuickLigase Buffer  33ul
       2x QuickLigase Buffer  33ul
       QuickLigase              1ul
       QuickLigase              1ul
       RT 15min -> purified with Qiaquick columns
       RT 15min -> purified with Qiaquick columns, eluted with 30ul EB.

Revision as of 00:37, 18 October 2009

Test the 1-adaptor ligation protocol

  • Billy sent me two tubes of sheared NA12878 samples captured by #6&7.
  • I did size selection on ~80ul of the sheared DNA from tube 1 using the SizeSelect gel, and extract ~100bp and ~120bp fragments.
  • I did End-repair on the extracted DNAs without further purification.
       DNA    45ul
       10X End-repair buffer    5.3ul
       Enzyme Mix               2.5ul
   25C for 45 min -> 70C for 10min -> purified with Qiaquick columns
   Ligation:
     Adaptor ligation: 20ul Solexa_1_up (200uM) + 20ul Solexa_1_lo_noP (200uM) 
          + 5ul 10X Stoffel buffer + 55ul H2O -> 94C 3min -> -0.1C/sec to 20C  
     DNA                     30ul
     40uM adaptors            3ul
     2x QuickLigase Buffer   34ul
     QuickLigase              1ul
     RT 15min -> purified with Qiaquick columns 
       -> extract 120bp+ and 150bp+ fragments with SizeSelect gel.
     PCR
     Template               5ul      5ul       5ul
     2x iProof Mix         50ul     50ul      50ul
     Solexa_PCR_up(100uM) 0.4ul    0.4ul     0.4ul
     AmpR6.3Sol(100uM)    0.4ul      -         - 
     AmpF6.3rSol(100uM)     -      0.4ul       - 
     50X SYBGI            0.4ul    0.4ul     0.4ul
     H2O                   44ul     44ul      44ul
     98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 -> (98C 10sec -> 72C 15sec) x 10 -> 72C 2min -> 15C hold.


  • I also did End-repair on another 80ul sheared DNA from tube 2 (prior to size selection).
       DNA                       85ul
       10X End-repair buffer     10ul
       Enzyme Mix                 5ul
       RT 30min -> 70C 10min -> purified with a Qiaquick column.
  • Perform size selection with SizeSelect gel, and extract ~120bp and ~140bp bands.
   Ligation:
     DNA                     30ul
     40uM adaptors            2ul
     2x QuickLigase Buffer   33ul
     QuickLigase              1ul
     RT 15min -> purified with Qiaquick columns, eluted with 30ul EB.