T3/T7 PRIMER QUESTION!!!

Darren Stauth stauth at crunch.usgmrl.ksu.edu
Thu Nov 3 12:19:51 EST 1994


I'm currently doing universal PCR to amplify and sequence an unknown region
of DNA.  I've used a couple of different universal primers, both of which
contain T7 primer sequence but found they caused problems because the TA
cloning vector also contains a T7 site.  This gives mixed sequence when
performing sequencing reactions with the T7 primer.  So we changed to a
uni-primer with a T3 site within it.  By some confusion I used T7 instead
of T3 in my second round of PCR yet still got amplification.  I assume
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



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