Bisulfite sequencing primer - (May/24/2006 )
If I were to design my sequencing primer with a degenerate base near the 5' end (can't get around one of the CpGs) would I be likely to somehow mess up the sequencing reaction?
Also I've been playing around with perl primer, but for whatever reason it never seems to give me a reverse sequencing primer based off of my parameters. Anyone else have this problem?
Tm = 60-65
Primer Length = 23-26
All options checked
Also, I'm a bit fuzzy on when it is possible to sequence with a single primer and when it is necessary to use both. Could anyone explain?
If you sequece after cloning, degenerate primer would not be a problem.
For direct sequencing, I usually only use the reverse primer which always gives better result than forward primer for unknown reason.
i would sequence from both ends if your insert exceeds the optimal length of sequence you get from DNA sequencing.
So a degenerate base in direct sequencing primer would not work?
can't see why not.