Kun:LabNotes/ExonomeSeq/2009-5-23

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Shotgun library construction

  • Start with four normalized and size-selected libraries captured from PGP1-3, NA12878 (prepared by Alice Li). The estimated concentration is ~2ng/ul each.

PCR with dUTP

                                      x 4 x 4
   Template:                   1ul
   2x EconoTaq Master Mix     50ul      800
   100uM AmpF6.3NH2          0.2ul      3.2
   100uM AmpR6.3NH2          0.2ul      3.2
   10mM dUTP                 0.6ul      9.6
   50x SYBG I                0.4ul      6.4
   H2O                        48ul      770
   94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C
   Purified every 4x50ul for each amplicon with two MinElute column.
 
   A: 23ng/ul x 20ul
   B: 14ng/ul x 20ul
   C: 14ng/ul x 20ul
   D: 18ng/ul x 20ul
   Purified each of the remaining amplicons with one DNA concentrator-5 column, eluted with 20ul EB
   A: 6.3ng/ul x 20ul
   B: 6.2ng/ul x 20ul
   C: 6.8ng/ul x 20ul
   D: 4.8ng/ul x 20ul

USER digestion

   DNA         20ul
   USER enzyme  2ul
   37C 1h

S1 nuclease digestion

 10 x S1 nuclease buffer:   3ul     
 DNA after USER digestion: 22ul    
 S1 nuclease (10U/ul):      2ul     
 ddH2O                      2ul     
 37C 15mins.
 Purified with MinElute cloumn. Elute in 16ul H2O.

End repair (Enzymatics End-repair Mix)

  Fragmented DNA  15ul
  dNTP           2.5ul
  10x buffer     2.5ul
  Enzyme mix       1ul
  H2O              4ul
  Incubate at RT for 30 minutes 
  Purified with Qiaquick column.
  Select the ~100bp fragments with 2% SizeSelect gel

Adaptor ligation(Enzymatic T4 ligase vs. NEB QuickLigase)

                            NEB     
  DNA                       20ul    
  2X Rapid Ligation buffer  25ul
  100uM Solexa_1 adaptor   0.5ul 
  100uM Solexa_2 adaptor   0.5ul   
  T4 DNA ligase(600U/ul)     1ul
  H2O                        3ul

Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns.

Nick translation

  DNA                  8ul
  10x ThermoPol buffer 1ul
  10mM dNTP          0.4ul
  Bst Pol (8U/ul)    0.5ul  
  65C 10min

PCR

                                  x2
  DNA                     10ul
  Solexa_PCR_up(10uM)      2ul
  Solexa_PCR_lo(10uM)      2ul
  2x iProof master mix    50ul
  50x SYBGI              0.4ul
  ddH2O                   36ul
  98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) 
  5 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold.