changing dNTP -> changing PCR conditions? - (Aug/18/2005 )

Hi!

Today I changed dNTP brand (but not concentration) in my optimized PCR reaction. And the most sensitive sample (very little DNA) gave a negative result, wheras I got a positive result on an easir sample.

Do the dNTP influence the result that much? Or was I just unlucky?
Or do I have to optimize the MgCl2-conc. again?

-k_josefin-

Try increasing the number of cycles that might help if your DNA conc is too less.

-Rishikesh-

QUOTE (Rishikesh @ Aug 18 2005, 03:39 PM)

Try increasing the number of cycles that might help if your DNA conc is too less.

Weel, I can't.. I'm doing a T-RFLP where only a small fraction of the samples are of low DNA concentration. And I want to use some kind of 'standard procedure' to be able to compare the samples (PCR biases increases with increasing number of PCR cycles)

-k_josefin-

Since dNTPs are prepared by two companies and just repackaged for all of the other suppliers, you have a 50% chance of having exactly the same dNTPs as before. There is no need to reoptimize the magnesium concentration if the template and primers are the same.
When the amount of DNA template is borderline, variations in the amount of template and enzyme sticking to the tube or well walls can lead to variable results. Adding a carrier such as tRNA or BSA can reduce this effect.

-tfitzwater-