Noi/NOTES/2011-9-13: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
No edit summary
>Noi
No edit summary
Line 1: Line 1:
= 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 of
* 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 (TTTTTTT read by reverse primer, Syb_RP7). One clone show only 6 T and another clone showed TTTCTTT. I am not sure if the last one is the random sequence from the survival AmpFNU6.3Sol 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 puul-up.
** 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)