PCR Using T7 and SP6 Primers

Eric Anderson e-anderson at ski.mskcc.arghhh
Thu Jul 31 08:30:03 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
>Ma

i routinely check inserts in pGEM with T7 and SP6 at an annealling temp of
55oC.  while i realize that this seems a bit high given the length and
sequence, i've been doing it this way for 5 years with no adverse effects.

as an example, if i want to PCR amplify an 800bp fragment in pGEM, my
conditions on a Perkin-Elmer 9600 machine are:

94oC-15s
55oC-15s
72oC-45s

25-30 cycles.

good luck,

eric

-- 
Eric C. Anderson
Sloan-Kettering Institute for Cancer Research 
Dept. of Cell Biology and Genetics
Lab: (212) 639-2977
Fax: (212) 717-3298
e-anderson at ski.mskcc.arghhh

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