PCR Using T7 and SP6 Primers
lha~ at med.pitt.edu
Thu Jul 31 11:27:07 EST 1997
In article <33DFE756.716C at ns.east.cn.net>, 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 check for plasmid inserts using PCR with the Forward and Reverse sequencing primers, we use a Tm of 52 C. We do "direct colony PCR", that is, prepare the master mix, aliquot it into PCR tubes, then simply pick a piece of the colony right off the agar plate with a loop and mix into into the mix, then cycle. Works every time. You can easily screen multiple colonies with one master mix.
The primers are: FOR: GTT TTC CCA GTC ACG ACG TTG TA
REV: TTG TGA GCG GAT AAC AAT TTC
Of course, check that these "universal" primers are present in your specific plasmid.
(Remove the tilde from my email if you need to email directly)
More information about the Methods