Kun:LabNotes/Haplotyping/2010-8-26

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Illumina multiplex library construction

  • Received sheared DNA from Sara.
  • End-repair:
   Fragmented DNA                              85 μl
   NEBNext End Repair Reaction Buffer (10X)    10 μl
   NEBNext End Repair Enzyme Mix                5 μl
                                               -----
                                              100 μl
   Incubate in a thermal cycler for 30 minutes at 20°C.
   Purified with AMPure beads.
  • A-Tailing:
   Blunt DNA                                42 μl
   NEBNext dA-Tailing Reaction Buffer (10X)  5 μl
   Klenow Fragment (3´→ 5´ exo–)             3 μl
                                            -----
                                            50 μl
   Incubate in a thermal cycler for 30 minutes at 37°C.
   Purified with AMPure beads.
  • Ligation:
   End Repaired, Blunt or dA-Tailed DNA     27 μl
   Quick Ligation Reaction Buffer (5X)       8 μl
   20 μM DNA Adaptors                      2.5 μl
   Quick T4 DNA Ligase                     2.5 μl
   Incubate in a thermal cycler for 15 minutes at 20°C. 
   Purified with AMPure beads.    
  • PCR:
  Adapter ligased DNA         10 μl
  PCR_F(10uM)                  2 μl
  PCR_R.N2IndX(10uM)           2 μl  
  50X SYBR Green I           0.8 μl
  H2O                         37 μl
  2X Phusion HF MM            50 μl
                              -----
                             100 μl
   Barcodes     200bp
   1A           Ind8
   1F           Ind9
   1G           Ind10
   2E           Ind11
   2F           Ind12
  98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min.
  Terminated the reactions before the curves reach the plateau stage.