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PCR detection

Gerri Newfry gnew at orion.it.luc.edu
Fri Jul 29 14:09:09 EST 1994


More PCR questions to the zen masters.

Is hot start really working better for folks?

I need to make a mutant, to include in my PCR reaction, so that I can 
quantitate the results.  What is the best way to detect the amplified 
mutant and amplified sample?  I was trying to detect the PCR products 
using fluoroscein conjugated UTP, or using 32-PdATP.  These methods were 
both unsuccessful, but I realize now that my basic PCR reaction wasn't 
working properly.  I don't 
like the 32-P because when I run the products on a gel, everything gets 
radioactive.  But I will use it if I have to.  Can anyone tell me how 
they've had success with FL-UTP?  Especially how you altered the 
concentration of the other dNTPs.

Do people freeze their primers?  I never have because we never freeze any 
of our DNA.

What is a generally useful dNTP concentration to begin with?  250 uM each?

Thanks in advance, and thanks to all those who replied to the zen of pcr post


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