PCR Using T7 and SP6 Primers

Ulrich Maier maieru at uni-muenster.de
Fri Aug 1 08:20:51 EST 1997

ddsyr at NS.EAST.CN.NET (ma) wrote:

>Dear Netters:
>I want to check the insert in plasmid (pGEM), so I plan to use a universe primers, 
>say T7(5'AAT ACG ACT CAC TAT AG 3') and SP6(5' ATT TAG GTG ACA CTA TAG 3'), which 
>are also used in DNA sequence. But I find the Tm of these two primers are very low, 
>only 42-45. So I guess this may influence these PCR efficiency. Hope the experts 
>give me their experiences and suggestions. Thanks

I have used the SP6/T7 sequencing primers from Pharmacia successfully
in PCR. The annealing temperature was 45°C, primer concentration 5pmol
per 50µl total volume. It´s quite expensive to use commercial
available sequencing primers for PCR.

If you ´ll choose another longer SP6-Primer for the amplification of
your insert, compare the primer with the vector sequence. The
conserved region of the SP6-site is really short, the flanking regions
are variable from vector to vector.


Ulrich Maier

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