Kun:LabNotes/GenomeSeq/2010-9-4

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Repeat the ligation PCR with barcoded and non-barcoded primers[edit]

  • Template: B.Jap-3.3
  • PCR:
  Adapter ligased DNA          1 μl  0                    1 μl   0
  PCR_F(10uM)                  2 μl  2                    2 μl   2
  PCR_R(10uM)                  2 μl  2   PCR_R_ind3(10uM) 2 μl   2
  50X SYBR Green I           0.8 μl .8                   .8 μl  .8
  H2O                         45 μl 45                   45 μl  45
  2X Phusion HF MM            50 μl 50                   50 μl  50
                              -----
                             100 μl
  98C 30sec -> (98C 10sec -> 66C 15sec -> 72C 15sec) x 18 cycles.
  File:2010-09-04-ligation-PCR.png
  • Conclusion:
    • The two negative controls were clean. Therefore the ~120bp bands are likely due to self-ligation of the adaptors.
    • I think I can simply repeat the amplification on the ligation products, and then do PAGE size selection to obtain the libraries of the desired sizes. Need to double check with Alice.