Jie:LabNotes/CpgSeq/2010-1-28

From ZhangLabWiki
Revision as of 20:57, 28 January 2010 by >Zsakura2 (Created page with '==seq lib with Harvard sample 7-10, Breast cancer sample 5-9== Harvard sample capture were performed on 2009-11-09 and 2009-11-20. NH2 reamplification were performed on 2010-01-…')
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

seq lib with Harvard sample 7-10, Breast cancer sample 5-9

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

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

                                       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

  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 -> 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.

I did the TBE gel size selection of ~250bp fragments of the seq libraries.