Kun:LabNotes/ExonomeSeq/2009-10-16

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Test the 1-adaptor ligation protocol

  • Billy sent me two tubes of sheared NA12878 samples captured by #6&7.

Batch 1

  • I did size selection on ~80ul of the sheared DNA from tube 1 using the SizeSelect gel, and extract ~100bp(L100) and ~120bp(L120) 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(10uM)    2ul      2ul       2ul
     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.

Batch 2

  • 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 (EL120) and ~140bp(EL140) bands.
   After extracting the DNA, I refilled each well with 20ul EB, ran for additional 3 minutes.
   File:2009-10-16 CloneWell-1.png
   Ligation:
     DNA                     30ul
     40uM adaptors            2ul
     2x QuickLigase Buffer   33ul
     QuickLigase              1ul
     RT 15min -> purified with Qiaquick columns, eluted with 30ul EB.
  • Perform PCR on a small fraction of the ligation products without size selection.
     PCR
     Template               2ul      2ul       2ul
     2x iProof Mix         50ul     50ul      50ul
     Solexa_PCR_up(10uM)    2ul      2ul       2ul
     AmpR6.3Sol(100uM)    0.4ul      -         - 
     AmpF6.3rSol(100uM)     -      0.4ul       - 
     50X SYBGI            0.4ul    0.4ul     0.4ul
     H2O                   45ul     45ul      45ul
     98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 10sec) x 8 
        -> (98C 10sec -> 72C 15sec) x 4 -> 72C 2min -> 15C hold.
   File:2009-10-17 ligation PCR.jpg
   A: Batch 1, 100bp
   B: Batch 1, 120bp
   C: Batch 2, 140bp
   D: Batch 2, 120bp
  • Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.
   File:2009-10-17-PAGE-sizeSelect.png