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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. dry.gif