Posted 12 December 2004 - 04:22 PM
I have isolated DNA from freshly isolated CD4+ T cells. The problem that I have is that I can find the expected amplicon (~400bp) in alternate PCRs...
The thing is after a PCR, no band is seen in the gel. When I repeat the reaction (same DNA, primers, & PCR conditions), the band appears, but not in the third, and so on...
How could this be explained? Sampling problems???
I will REALLY appreciate your suggestions...
Posted 12 December 2004 - 06:33 PM
(you think i'm kidding...i'm not).
do you run a positive control?
do you mix the reaction *really* well beforehand?
are all of the bits and pieces unfrozen before putting them in the reaction?
seriously, try to figure out what is different with the reactions that work, and those that don't. did you stick things in the tube in the same order?
if there is really nothing different, then, a, it's the magic trolls, b, it's the position of solar flares, and c, it's the moon's influence over the cosmic relation of stuff.
with the amounts that are involved in a PCR reaction, it only takes a little bit to be out for the entire reaction to flop.
Edited by vetticus3, 12 December 2004 - 06:59 PM.
Posted 12 December 2004 - 07:40 PM
Just a thought... have you actually checked to see that the product is the amplicon you want? It could be that there is some random contaminant in one of the reagents, that is coming out in seemingly every second PCR.
My other suggestion is to try fresh primers and dNTPs, sometimes when these degrade PCR does funny things. I too have had problems similar to yours, and the solution was fresh primers and dNTPs.