Kun:LabNotes/GenomeSeq/2010-9-4: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
m (Created page with '==Repeat the ligation PCR with barcoded and non-barcoded primers== *Template: B.Jap-3.3 *PCR: Adapter ligased DNA 1 μl 0 1 μl 0 PCR_F(10uM)…')
 
 
Line 6: Line 6:
   PCR_R(10uM)                  2 μl  2  PCR_R_ind3(10uM) 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
   50X SYBR Green I          0.8 μl .8                  .8 μl  .8
   H2O                        37 μl 38                   37 μl  38
   H2O                        45 μl 45                   45 μl  45
   2X Phusion HF MM            50 μl 50                  50 μl  50
   2X Phusion HF MM            50 μl 50                  50 μl  50
                               -----
                               -----
                               100 μl
                               100 μl
   98C 30sec -> (98C 10sec -> 68C 15sec -> 72C 15sec) x 18 cycles.
   98C 30sec -> (98C 10sec -> 66C 15sec -> 72C 15sec) x 18 cycles.
  [[Image:2010-09-04-ligation-PCR.png|300px]]
*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.

Latest revision as of 21:15, 4 September 2010

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.