Anyone having problems with PCR Blunt?

Anthony Anthony
Thu Feb 6 11:49:36 EST 1997


In article <5c0m00$7ff at net.bio.net> Suzanne McNeely,
smcneely at sunstroke.sdsu.edu writes:
>Invitrogen recently came out with a new plasmid called PCR Blunt. One of
>our investigators has just tried it in autosequencing and we are having
>dismal results. We sequence with T7 and Reverse primers. The problem with
>T7 is that we get nothing, and I do mean nothing. I can't even scare
>something up by playing with the raw data.  With reverse we get really
>beautiful data up to the insert then bam!,  nothing. We've had the
>investigator take some of the plasmid back and try to cut out the insert
>just to ensure it's really there and he gets a bright beautiful single
>band. I'm inclined to believe this must be a problem with the plasmid. Is
>anyone else out there sequencing this plasmid and how are the results? Any
>other possibilities I may be missing? Note: These primers work great on
>other samples on the same gel.

Is it possible that it's your insert?  If it has very strong 2ndary
structure, it
might just block the pol completely.  Maybe if you tried sequencing the
excised
insert with an internal primer you could answer this question.  Also,
have you
tried empty PCR Blunt, or with a different insert?

Anthony.

P.S. Just being Devil's advocate!  I'm sure PCR Blunt might well be crap;
the name 
certainly is.



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