Noi/NOTES/2011-9-13: Difference between revisions
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= Sanger sequencing results of the amplicons amplified by randomly tagging primers with and without USER = | = Sanger sequencing results of the amplicons amplified by randomly tagging primers with and without USER = | ||
* 10 clones | * 10 clones from the reaction with USER --> if there is no AmpFNU.Sol primer left over after USER digestion, we should observe every clone contains poly T sequences (TTTTTTT -> 7T) by reading with reverse primer, Syb_RP7. | ||
** 8 clones showed very clear poly T sequences | ** 8 clones showed very clear poly T sequences. One clone showed only 6 T and another clone showed TTTCTTT. I am not sure if the second one was derived from the random sequences of the AmpFNU.Sol primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the comment of the this sample result, it said that there was a spectral pull-up that might be caused from too much of DNA in the reaction. | ||
[[File:NP-NA-U-01.png| 600px]]<br> | [[File:NP-NA-U-01.png| 600px]]<br> | ||
[[File:NP-NA-U-09.png| 600px]]<br> | [[File:NP-NA-U-09.png| 600px]]<br> | ||
* 10 clones of positive control with no USER | * 10 clones of positive control with no USER --> we expected to see the mix of polyT (TTTTTTT -> 7T) and random sequences (NNNNNNN -> 7N) | ||
** 4 clones with poly T | ** 4 clones with poly T (TTTTTTT -> 7T) | ||
** 6 clones with random sequences (NNNNNNN) | ** 6 clones with random sequences (NNNNNNN -> 7N) |
Revision as of 00:04, 14 September 2011
Sanger sequencing results of the amplicons amplified by randomly tagging primers with and without USER
- 10 clones from the reaction with USER --> if there is no AmpFNU.Sol primer left over after USER digestion, we should observe every clone contains poly T sequences (TTTTTTT -> 7T) by reading with reverse primer, Syb_RP7.
- 8 clones showed very clear poly T sequences. One clone showed only 6 T and another clone showed TTTCTTT. I am not sure if the second one was derived from the random sequences of the AmpFNU.Sol primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the comment of the this sample result, it said that there was a spectral pull-up that might be caused from too much of DNA in the reaction.
File:NP-NA-U-01.png
File:NP-NA-U-09.png
- 10 clones of positive control with no USER --> we expected to see the mix of polyT (TTTTTTT -> 7T) and random sequences (NNNNNNN -> 7N)
- 4 clones with poly T (TTTTTTT -> 7T)
- 6 clones with random sequences (NNNNNNN -> 7N)