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

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
mNo edit summary
>Sam Chiang
Line 1: Line 1:
==Test the 1-adaptor ligation protocol==
==Test the 1-adaptor ligation protocol==
*Billy sent me two tubes of sheared NA12878 samples captured by #6&7.  
*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 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.
*I did End-repair on the extracted DNAs without further purification.
         DNA    45ul
         DNA    45ul
Line 21: Line 21:
       Template              5ul      5ul      5ul
       Template              5ul      5ul      5ul
       2x iProof Mix        50ul    50ul      50ul
       2x iProof Mix        50ul    50ul      50ul
       Solexa_PCR_up(100uM) 0.4ul   0.4ul    0.4ul
       Solexa_PCR_up(10uM)    2ul      2ul      2ul
       AmpR6.3Sol(100uM)    0.4ul      -        -  
       AmpR6.3Sol(100uM)    0.4ul      -        -  
       AmpF6.3rSol(100uM)    -      0.4ul      -  
       AmpF6.3rSol(100uM)    -      0.4ul      -  
Line 35: Line 35:
         Enzyme Mix                5ul
         Enzyme Mix                5ul
         RT 30min -> 70C 10min -> purified with a Qiaquick column.
         RT 30min -> 70C 10min -> purified with a Qiaquick column.
*Perform size selection with SizeSelect gel, and extract ~120bp and ~140bp bands.
*Perform size selection with SizeSelect gel, and extract ~120bp (EL120) and ~140bp(EL140) bands.
     Ligation:
     Ligation:
       DNA                    30ul
       DNA                    30ul
Line 42: Line 42:
       QuickLigase              1ul
       QuickLigase              1ul
       RT 15min -> purified with Qiaquick columns, eluted with 30ul EB.
       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
*Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.

Revision as of 01:06, 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(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.


  • 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.
   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
  • Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.