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Reamplification of RT-PCR product - (Jun/17/2005 )

I got a good band after RT-PCR (touchdown PCR).But when I took 1micro liter out of this amplified product as a template for 25 micro liter PCR reaction I could not get any band though all other conditions were same.
What might be the reason? Should I increase or decrease the volume of the template or change the programming of touchdown PCR?
Kindly reply.


I couldn't see any reason why you could not get re-amplification. Nothing was wrong.


Purify your product prior to re-pcring it. Any carryover, other then template will screw up susequent reactions.