Kun:LabNotes/GenomeSeq/2010-9-4: Difference between revisions
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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)…') |
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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 | 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 -> | 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.