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

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m (New page: ==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 Size...)
 
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*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 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.
*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
      2x Kapa Mix          50ul    50ul
      Solexa_PCR_up(100uM) 0.4ul    0.4ul
      AmpR6.3Sol(100uM)    0.4ul      -
      AmpF6.3rSol(100uM)    -      0.4ul
      H2O                  44ul    44ul
      95C 30sec -> (95C 3sec -> 60C 30sec) x 18 -> 60C 2min -> 15C hold.
     
       
*I also did End-repair on another 80ul sheared DNA from tube 2 (prior to size selection).
*I also did End-repair on another 80ul sheared DNA from tube 2 (prior to size selection).

Revision as of 01:17, 17 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
     2x Kapa Mix           50ul     50ul
     Solexa_PCR_up(100uM) 0.4ul    0.4ul
     AmpR6.3Sol(100uM)    0.4ul      -
     AmpF6.3rSol(100uM)     -      0.4ul
     H2O                   44ul     44ul
     95C 30sec -> (95C 3sec -> 60C 30sec) x 18 -> 60C 2min -> 15C hold.



  • I also did End-repair on another 80ul sheared DNA from tube 2 (prior to size selection).