Smeary touchdown PCR gel.

[Stephanie Yong]

Dear Netters,

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
55 C,30sec),END.

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.
Stephanie.