Jie:LabNotes/CpgSeq/2010-1-21
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seq lib with Harvard sample 1-6[edit]
capture were performed on 2009-11-09 and 2009-11-20. NH2 reamplification were performed on 2010-01-06.
endrepair with NEB end-repair kit[edit]
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[edit]
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[edit]
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[edit]
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.jpgHarvard sample1~6 seq lib image
I did the TBE gel size selection of ~250bp fragments of the seq libraries. File:20100122 Harvard sample 1~6 seq lib TBE size selection.jpg20100122_Harvard sample 1~6 seq lib TBE size selection
I gave H_1, H_2, H3 for sequencing HL035. Labelling as follows: H1: JD-cpg97k-Harvard_sample-B0047.1_Jan22.2010 H2: JD-cpg97k+30k-Harvard_sample-B0060.1_Jan22.2010 H3: JD-cpg97k-Harvard_sample-B0070.1_Jan22.2010
I gave H_4, H_5, H6 for sequencing HL036. Labelling as follows: H4: JD-cpg97k-Harvard_sample-B0107.1_Jan22.2010 H5: JD-cpg97k-Harvard_sample-B0108.1_Jan22.2010 H6: JD-cpg97k-Harvard_sample-B0134.1_Jan22.2010