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what's wrong with my 3' RACE - (Aug/21/2006 )

ph34r.gif I've been stuck with my 3'race for a long time.currently, I use the smart race kit from BD. I did it according to the protocal, but after the 1st round pcr, there was no band---only smear. So I turned to the 2rd nest pcr. This time, it seemed that there were several bands ,but not so clear.
Attached Image 1st round pcr
Attached Image 2rd nest pcr
I'm not sure if I have to start again(extracting RNA------3'RACE), or set a series of pcr reaction using the 1st pcr product as template. sad.gif

-donandken-

for my 3'RACE i often took 1ul of the first round PCR- diluted it 1:100 and redid the pcr....seemed to get better bands (but my smear wasn;t quite as bright as yours) - how many cycles have you been doing?

-aussieuk-

QUOTE (aussieuk @ Aug 22 2006, 05:22 PM)
for my 3'RACE i often took 1ul of the first round PCR- diluted it 1:100 and redid the pcr....seemed to get better bands (but my smear wasn;t quite as bright as yours) - how many cycles have you been doing?

30 cycle

-donandken-