T3/T7 PRIMER QUESTION!!!
mkennedy at chmeds.ac.nz
Tue Nov 8 21:25:38 EST 1994
In article <stauth-031194111951 at darenmac.usgmrl.ksu.edu>, stauth at crunch.usgmrl.ksu.edu (Darren Stauth) writes:
> since T7 and T3 are so similar that T7 misannealed to the T3 site. This
> leads me to my question: since T7 and T3 are so similar and can misanneal
> to give amplification in PCR, will the same thing happen in sequencing
> reactions? I've seen vectors containing both T7 and T3 sites. Has anybody
> had problems sequencing in such a vector?
> Darren M. Stauth -- stauth at crunch.usgmrl.ksu.edu
I've never noticed mispriming, and I use T3/T7 a lot on Bluescript plasmids.
I must admit, until you mentioned it I'd never noticed these things are
quite similar, especially at their 3' ends:
T3 primer 5'-ATTAACCCTCACTAAAG-3'
| ||||||| ||
T7 primer 5'-AATACGACTCACTCACTATAG-3'
With other primers I have noticed how a single mismatch will screw sequencing,
but not affect PCR, so sequencing (at least with T7 polymerase) is more
sensitive to subtle differences between template and primer than PCR.
NNNN NN Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz) ZZZZZZZ
NN NN NN Cytogenetic and Molecular Oncology Unit ZZZ
NN NN NN Christchurch School of Medicine ZZZ
NN NNNN Christchurch, New Zealand ZZZZZZZ
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