Best primers for ABI sequencing of Bluescript

(Ian Ridgers) ir at nhm.ac.uk
Tue Aug 29 08:45:07 EST 1995


On Wed, 23 Aug 1995 16:17:11 +010,
  RM Brownlie writes:

>I have inserts cloned into the EcoRI/XhoI site of the Bluescript II SK 
>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

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 ir at nhm.ac.uk
Tel. 0171 938 9297

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