RACE sequencing problem
(by SBrown from ccia.unsw.edu.au)
Fri Dec 19 12:38:40 EST 2008
I have been sequencing constructs about 1kb in size that i have obtained from a second race reaction, i have pruified the product, spec'd it and ensured when run on a gel i get a band.
This has worked for a while, however the last 1 or so constructs i have had sequenced have failed, the chromatogram looks as though the primer has not even bound to the template, it is simply noise that drops off to an almost undetectable signal.
Can anyone make any suggestions as to what I should be looking at to troubleshoot this? I sent my positive control as well as 1 sample from my next set of rave reactions to be sequenced, both were successful, then when i sent the remaining 8, all 8 failed.
Might i need to redesign my primer i am using for sequencing? Any other thoughts?
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