Best primers for ABI sequencing of Bluescript
RM Brownlie
vector and would like to know which are the best primers for Dye
Terminator cycle sequencing using an ABI automated sequencer. Perkin and
Elmer recommend the M13 forward and reverse primers but the reverse
primer is a considerable distance from the EcoRI site. Has anyone had
much success using the T7 or T3 standard primers or modifications of and
did they modify any of the standard conditions? I would also like to know
whether it is worth doing single strand rescue to obtain maximum read
lengths?
Any information regarding achieving optimum results would be gratefully
received.
Robert Brownlie
Ian Ridgers
Robert
I didn't see any responses to your mail. However, for the record I also
have inserts in Bluescript. I was using the SK primer, which was giving
reasonably good results. But PE suggested I use the various other primers
instead. I've used M13 reverse, but like you said it's along way from the
ECoRI site, and it seems such a waste sequencing 130 bases of plasmid. I'm
now using T3 and T7 and getting very nice results (450 bases). Because of
their distance from the insert any excess dye terminator blobs etc occur in
the region where the plasmid sequence exists (?). I haven't changed any of
the conditions in the cycle, although PE did suggest altering the
extension time and doing hot starts.
Ian
Ian Ridgers
Dept. of Zoology
The Natural History Museum
Cromwell Rd.
London. SW7 5BD
E-mail i...@nhm.ac.uk
Tel. 0171 938 9297
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