Thu Nov 10 15:42:12 EST 1994

In article <stauth-031194111951 at darenmac.usgmrl.ksu.edu>
stauth at crunch.usgmrl.ksu.edu (Darren Stauth) writes:
>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
I have been using T7 and T3 as forward and reverse primers for amplification
of inserts in lambda ZapII without any problem.  I didn't think to try it with
just a single primer to see if one could bind to both sites.  That would be
an interesting possible source of problem with our direct sequencing of the
PCR products.  I have seen sequence that looked like two different things
being sequenced at once in about 25% of my reactions.
Stuart Brown                       |   Plant Genetic Resources
                                   |   Georgia Experiment Station
INTERNET:                          |   1109 Experiment Street
  SBROWN at GAES.GRIFFIN.PEACHNET.EDU |   Griffin, Georgia 30223-1797 USA
BITNET:   SBROWN at GRIFFIN           |   Fax: 404-229-3323

More information about the Methods mailing list