Kun:LabNotes/ExonomeSeq/2009-10-16: Difference between revisions
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95C 30sec -> (95C 3sec -> 60C 30sec) x 18 -> 60C 2min -> 15C hold. | 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). | ||
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 |
Revision as of 01:20, 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).
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