Smeary touchdown PCR gel.
spyong at pc.jaring.my
Wed Aug 14 04:48:25 EST 1996
I have just received my degenerate primers and tried the Touchdown method
for PCR.We have the Perkin Elmer 2400 and it has a pre-programmed Touchdown
PCR protocol. I ran my samples using that protocol.
The programme :94 C,30sec,(94 C,30sec and 65 C,30sec) for 20 cycles
decreasing temp by 0.5 C till 55 C then it has a 10 cycle of (94 C,30sec and
My prep contain : 30pmol each primers, 1.5mM Mg, 5%DMSO,10mM mix dNTPs, 1.5U Taq
Total vol:25 microlitre.
I did an RT ,then diluted these for my PCR.
My Tm for the primers are 60 C. I was told very briefly, that if the temp is
below 70 C, a two -step prog. is relevant, and that I have to use the three-
step as my primers have a Tm below 70.Is this true?
Please could anyone be so kind as to elaborate on this.
My email: spyong at pc.jaring.my
Thanking all in advance.
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