Jie:LabNotes/CpgSeq/2010-1-21: Difference between revisions
Jump to navigation
Jump to search
>Zsakura2 (Created page with '==seq lib with Harvard sample 1-6== ===endrepair with NEB end-repair kit=== endrepair with NEB end-repair kit x6 85 ul DNA 10 ul 10X End…') |
>Jie deng No edit summary |
||
Line 39: | Line 39: | ||
98C 30sec-> 7 cycles of (98C 10sec->65C 20sec->72C 20sec)->72C 3 mins. | 98C 30sec-> 7 cycles of (98C 10sec->65C 20sec->72C 20sec)->72C 3 mins. | ||
I did the | [[Image:20100121_Harvard sample1~6 seq lib PCR curve.png|300px]]Harvard sample1~6 seq lib PCR curve | ||
[[Image:20100121_Harvard sample1~6 seq lib image.png|300px]]Harvard sample1~6 seq lib image | |||
I did the TBE gel size selection of ~250bp fragments of the seq libraries. |
Revision as of 18:05, 22 January 2010
seq lib with Harvard sample 1-6
endrepair with NEB end-repair kit
endrepair with NEB end-repair kit x6 85 ul DNA 10 ul 10X End-Repair Buffer 60 5 ul End-Repair Enzyme Mix 30 100 ul Total reaction volume 20C for 30 min. Purify with AMPureXP bead. Elute in 37ul elution buffer.
A tail addition
x6 Blunt-ended DNA 37ul 10X dA-tailing buffer 5ul 30 Klenow fragment (exo-)10U/ul 3ul 18 H2O 5ul 37C 30min, purified with beads, eluted with 40ul EB.
Ligation
I set up a negative control from this step. Adaptor ligation: A_adaptor_b(100uM) 20ul + A-adaptor_t (100uM) 20ul + H2O 60ul. -> 94C 3min -> -0.1C/sec to 20C x7 DNA 32ul 20uM adaptors 3ul 21 5x QuickLigase Buffer 10ul 70 Quick T4 Ligase 5ul 35 20C 15min -> I took 20ul for bead purification. Elute in 40ul buffer.
PCR after bead purification
I took 5ul purified products for PCR. Template 5ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 4ul Solexa_PCR_lo_PE (10uM) 4ul H2O 40ul 50X SYBG I 0.4ul 98C 30sec-> 7 cycles of (98C 10sec->65C 20sec->72C 20sec)->72C 3 mins. File:20100121 Harvard sample1~6 seq lib PCR curve.pngHarvard sample1~6 seq lib PCR curve File:20100121 Harvard sample1~6 seq lib image.pngHarvard sample1~6 seq lib image
I did the TBE gel size selection of ~250bp fragments of the seq libraries.