PCR my cDNA & got nothing - (Apr/13/2006 )

I got my cDNA which is 1.5kb using Qiagen's Onestep RT-PCR kit two months ago. I used gene-specific primers.

Now I want to clone it into pUC19, but find out cDNA is not enought. So I tried to PCR my cDNA.

I used NEB's Phusion polymerase with its buffer (Mg2+=1.5mM). cDNA template 1 micorliter (10ng). Annealing temp. 55 degree (Tm=61-62 degree). Primers were the same as in RT-PCR before. 30 cycle with no bands on gel.

Then I raised Mg2+=2.5mM. Increase template to 6 microliters (60ng). Still got nothing.

Finally, I give up Phusion and change to Promega's goTaq Green. But still no bands.

By the way, the cDNA is ok when I ran it as a control on gel.

Anyone have any idea can help me out? Thanks.

-tombyuan-

have you had degradation of the ends, so the primers will not work? I only ask because of the time lapse...?

-aimikins-

QUOTE (aimikins @ Apr 14 2006, 01:28 PM)

have you had degradation of the ends, so the primers will not work? I only ask because of the time lapse...?

I'm not sure. My primers are 30nt including RE site near both ends. I kept the cDNA in 4 degree in EB buffer for a month. Shouldn't degrade just in the ends. I've checked the cDNA and it has the right size.

-tombyuan-