PCR mixing - First tubes generally work, last ones generally don't. (May/28/2006 )

Hi,

I have a recurring problem in my PCR. It seems like most of the time when I do a PCR, the first few tubes that i filled always work, and the last ones that i did don't work. This happens even when i load my DNA straight into each tube.

I thought this was a problem with my mixing, so now I make sure to pump my pipette up and down when i am a) getting buffer, etc out of their containers, and when i divide the master mix between tubes.


SHould i be vortexing all the 'ingredients' before adding them?

ANd could the fact that i wasn't doing this earlier mean that something is now wrong with my stocks?

Any help greatly appreciated

its very frustrating

hi gstar
just wondering if you are making a master mix ? - if so, try to make 1.5X what you will actually need as it is possible that you are running out towards the end - could this be it?
cheers

yeah, mixing each component before adding to the master mix is good, as things can settle out

but here's another question, just to be sure we're barking up the right tree: you don't usually do the same number of samples, and put them in the block the same way? if it is related to spacial orientation in your block, perhaps your thermalcycler's cranking out on you?

Thanks for your replies

Say I am doing 8 tubes, I usually make up enough mix for 9 and this doesn't seem to run out, so I don't think this would be the case. Usually only the first one or two tubes work and the rest don't work at all

Aimikins, you have an interesting point - although I do use different numbers of samples, I guess the first ones usually go in around about the same spot. We do have a machine that does gradients so I might just check that they aren't set up..


Thanks again