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