Kun:LabNotes/GenomeSeq/2010-8-27

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Repeat the Illumina library construction on B.Jap3.3 & B.Jap3.8[edit]

  • End-repair:
   Fragmented DNA                              30 μl
   NEBNext End Repair Reaction Buffer (10X)   3.5 μl
   NEBNext End Repair Enzyme Mix              1.5 μl
                                               -----
                                              35 μl
   Incubate in a thermal cycler for 30 minutes at 20°C.
   Purified with MinElute columns, eluted with 15ul EB
  • A-Tailing:
   Blunt DNA                                14.5 μl
   NEBNext dA-Tailing Reaction Buffer (10X)    2 μl
   Klenow Fragment (3´→ 5´ exo–)             1.5 μl
   H2O                                         2 μl
                                            -----
                                            20 μl
   Incubate in a thermal cycler for 30 minutes at 37°C.
   Purified with MinElute columns, eluted with 15ul EB
   Conc(ng/ul)  300bp       800bp 
   B.Jap-3       43          56

  • Ligation:
                10:1         20:1
   Adaptors     300bp        800bp 
   B.Jap-3      1ul           1ul
   B.Jap-3(old) 1ul           1ul
   End Repaired, Blunt or dA-Tailed DNA     13 μl
   Quick Ligation Reaction Buffer (5X)       4 μl
   20 μM DNA Adaptors                        1 μl
   Quick T4 DNA Ligase                     1.5 μl
   H2O                                     0.5 ul
   Incubate RT for 15 minutes. 
   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     300bp       800bp  
   B.Jap-3      Ind3        Ind9 
 
 98C 30sec -> (98C 10sec -> 68C 15sec -> 72C 15sec) x 15 cycles.
 Terminated the reactions at 10th cycle.
 File:2010-08-28-ligation-PCR.png
  • Conclusion:
    • The ~120bp band still existed, indicating that it has nothing to do with end-polishing reactions and ligations.