Noi/NOTES/2011-9-13: Difference between revisions
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* Continued from 2011_09_07: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-7#Ligation_set_up]]<br> | * Continued from 2011_09_07: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-7#Ligation_set_up]]<br> | ||
* 10 clones from the reaction with USER --> if there was 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. | * 10 clones from the reaction with USER --> if there was 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 (7T). One clone (NP-NA-U-01) showed only 6T and another clone (NP-NA-U-09) showed TTTCTTT instead of TTTTTTT. For the first one, I think that it should be the error of primer synthesis since the surrounded sequences were corrected ('''GAGAGTG'''TTTTTT'''GTGTAGA'''). For the second one, I am not sure if it derived from the '''AmpFNU.Sol''' primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the failure cause of the this sample, it said that there was a spectral pull-up that might be caused by too much DNA in the reaction. For me, I believed that it should | ** 8 clones showed very clear poly T sequences (7T). One clone (NP-NA-U-01) showed only 6T and another clone (NP-NA-U-09) showed TTTCTTT instead of TTTTTTT. For the first one, I think that it should be the error of primer synthesis since the surrounded sequences were corrected ('''GAGAGTG'''TTTTTT'''GTGTAGA'''). For the second one, I am not sure if it derived from the '''AmpFNU.Sol''' primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the failure cause of the this sample, it said that there was a spectral pull-up that might be caused by too much DNA in the reaction. For me, I believed that it should derive from the AmpAU.Sol primer as the reason of spectral pull-up that T pulled up G, C then A. | ||
[[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> |
Revision as of 19:03, 14 September 2011
Sanger sequencing results of the amplicons amplified by randomly tagging primers with and without USER
- Continued from 2011_09_07: [[1]]
- 10 clones from the reaction with USER --> if there was 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 (7T). One clone (NP-NA-U-01) showed only 6T and another clone (NP-NA-U-09) showed TTTCTTT instead of TTTTTTT. For the first one, I think that it should be the error of primer synthesis since the surrounded sequences were corrected (GAGAGTGTTTTTTGTGTAGA). For the second one, I am not sure if it derived from the AmpFNU.Sol primer or the artifact of sequencing since the DNA chromatogram showed a very clear C peak. However, based on the failure cause of the this sample, it said that there was a spectral pull-up that might be caused by too much DNA in the reaction. For me, I believed that it should derive from the AmpAU.Sol primer as the reason of spectral pull-up that T pulled up G, C then A.
File:NP-NA-U-01.png
File:NP-NA-U-09.png
- 10 clones of positive control with no USER in the reaction --> 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)