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