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