Jie:LabNotes/CpgSeq/2010-1-28
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seq lib with Harvard sample 7-10, Breast cancer sample 5-9[edit]
Harvard sample capture were performed on 2009-11-09 and 2009-11-20. NH2 reamplification were performed on 2010-01-06. Breast cancer sample
endrepair with NEB end-repair kit[edit]
endrepair with NEB end-repair kit x9 85 ul DNA 10 ul 10X End-Repair Buffer 90 5 ul End-Repair Enzyme Mix 45 100 ul Total reaction volume 20C for 30 min. Purify with AMPureXP bead. Elute in 37ul elution buffer.
A tail addition[edit]
x9 Blunt-ended DNA 37ul 10X dA-tailing buffer 5ul 45 Klenow fragment (exo-)10U/ul 3ul 27 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 x10 DNA 32ul 20uM adaptors 3ul 30 5x QuickLigase Buffer 10ul 100 Quick T4 Ligase 5ul 50 20C 15min -> bead purification. Elute in 40ul buffer.
PCR after bead purification[edit]
I took 2.5ul purified products for PCR. Do 200ul for each sample Template 2.5ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 2ul Solexa_PCR_lo_PE (10uM) 2ul H2O 40ul 50X SYBG I 0.4ul 98C 30sec-> 7 cycles of (98C 10sec->65C 20sec->72C 20sec)->72C 3 mins. I did the TBE gel size selection of ~250bp fragments of the seq libraries.
File:20100128 Harvard and Breast cancer sample seq lib PCR.png20100128_Harvard and BCS seq lib PCR
File:20100128 Harvard and Breast cancer sample seq lib.jpg20100128_Harvard and BCS seq lib quantification
I did the gel quantification and give samples for sequencing: The following 4 samples are sequenced on HL038: H7: JD-cpg97k-Harvard_sample-M0013.1_Jan28.2010 12.55ng/ul H8: JD-cpg97k-Harvard_sample-M0077.3_Jan28.2010 3.47ng/ul H9: JD-cpg97k-Harvard_sample-M0124.3_Jan28.2010 5.64ng/ul H10: JD-cpg97k-Harvard_sample-M0188.3_Jan28.2010 3.75ng/ul
The B5 and B6 were sequencd on s2 and s3 of 100207_HL038. B5: JD-cpg97k-Breastcancer_sample-01436B09_Jan28.2010 6.36ng/ul B6: JD-cpg97k-Breastcancer_sample-01523B09_Jan28.2010 5.55ng/ul
The B7 and B8 were sequenced on s5 and s6 of 100212_HL039. B7: JD-cpg97k-Breastcancer_sample-03170B09_Jan28.2010 5.97ng/ul B8: JD-cpg97k-Breastcancer_sample-03259B09_Jan28.2010 6.69ng/ul