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

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*Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.
*Perform PAGE size selection on ~24ul of the ligation products in 2 wells of the 5-well TBE gel.
     [[Image:2009-10-17-PAGE-sizeSelect.png|400px]]
     [[Image:2009-10-17-PAGE-sizeSelect.png|400px]]
*Perform PCR on the PAGE size selected DNA:
      PCR                  F+R1  F+R1+R2 
      Template              5ul      5ul   
      2x iProof Mix        50ul    100ul   
      Solexa_PCR_up(10uM)    2ul      4ul     
      AmpR6.3Sol(100uM)    0.2ul    0.4ul     
      AmpF6.3rSol(100uM)    -      0.4ul     
      50X SYBGI            0.2ul    0.4ul   
      H2O                  45ul    90ul   
      98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 20sec) x 10 -> 72C 2min -> 15C hold.
    [[Image:2009-10-19-ligation-PCR.png|300px]]
==Prepare amplicons for sequencing==
*Pooled the Batch 2 amplicons that were amplified from non-size-select ligation products with two primer sets. Labeled them EL-120.2 & EL140.2, purified with Qiaquick columns.
*Labeled the amplicons that were amplified from size-selected ligation products using the three primers with EL-120.3, EL140.3. Purified with Qiaquick columns.


==Conclusion==
==Conclusion==

Revision as of 21:57, 19 October 2009

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                   F+R1     F+R2       F 
     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                   F+R1     F+R2       F 
     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
  • Perform PCR on the PAGE size selected DNA:
     PCR                   F+R1   F+R1+R2  
     Template               5ul      5ul     
     2x iProof Mix         50ul    100ul     
     Solexa_PCR_up(10uM)    2ul      4ul      
     AmpR6.3Sol(100uM)    0.2ul    0.4ul       
     AmpF6.3rSol(100uM)     -      0.4ul      
     50X SYBGI            0.2ul    0.4ul     
     H2O                   45ul     90ul     
     98C 30sec -> (98C 10sec -> 60C 20sec -> 72C 20sec) x 10 -> 72C 2min -> 15C hold.
   File:2009-10-19-ligation-PCR.png

Prepare amplicons for sequencing

  • Pooled the Batch 2 amplicons that were amplified from non-size-select ligation products with two primer sets. Labeled them EL-120.2 & EL140.2, purified with Qiaquick columns.
  • Labeled the amplicons that were amplified from size-selected ligation products using the three primers with EL-120.3, EL140.3. Purified with Qiaquick columns.

Conclusion

  • The one-adaptor protocol worked.
  • After end-repair, size selection can be performed with SizeSelect gel, which is fast and easy. The fragments to select should center around 120bp.
  • No size selection is necessary on the ligation products prior to PCR.
  • The ligation PCR amplicons seem to be clean enough to be sequenced directly without further size selection.