Buffer-composition One-Step RT-PCR - (Jul/11/2012 )
in our lab we're using Promegas One-Step RT-PCR System Access. But we encountered a problem with the buffer supplied with the kit. The buffer supplied is really tight calculated and so we usually run out of buffer before we use up all the supplied enzyms, so we fall short around 20-30 rxns.
Does anybody know the composition of the 5x AVM/Tfl reaction buffer? I looked up the composition of the individual buffers for AVM-RT and Tfl-Pol, but they don't match of course.
Any help is much appreciated, as Promega Customer Service is not to helpful to sell or provide us with extra reaction-buffer, and we would like to avoid to adjust all our assays to another kit.
It's difficult. it would need lots of optimization. I have buffer components for normal PCR, but for RT I am not really sure.
That is one of the reasons I don't like one-step RT-PCR....can't you just synthesize cDNA and then use it in normal PCR? Maybe you can ask around your lab if anyone has cDNA synthesis kit?
Also, I have used Promega's access kit, and it works quite well. I never ran out of the buffer! why does this happen to you?
Thanks Curtis for taking part in this discussion. Well, we could synthesize regularly cDNA, we have the enzymes, but we prefer not to. The Access kit is used for diagnostic samples, so for higher sensitivity and less contamination as well as easier handling when having a lot of samples. I really don't know why we run out of buffer first. All people in the lab have several years of experience in molecular work, and I don't think it's some kind of pipetting error.
Any suggestion on how to identify the mistake we're making?
This does seem very strange. I know for most kits I've ever used (granted I haven't used a one step rt pcr kit though...) buffers are supplied in a slight excess to the enzyme or whatever.
Is it at all possible that your pipette itself is out and needs calibration? What kind of volumes are we talking here?
The pipette was calibrated and should be fine. I talked to the support and they said the buffer tube is slightly overfilled. So for on rxn you need to use 10 ul of buffer. The mastermix are usually between 4 and 13 rxns, so 130 ul tops.
When summarizing the answers we should think about recalibrating the pipette.