Real-time PCR problems - RT_PCR (Feb/06/2006 )

I am trying to get our RT-PCR machine up and running so that I can graduate sooner than later. With that being said, any help would be greatly appreciated.

We are trying to see any sort of amplification. Our reactions are 20 uL in capillaries with 2 uL of SYBR Green Master Mix, 2 uL DNA, different amounts of primers, and filling to a final volume with nuclease-free water. The PCR protocol we have is from the literature on very similar primers and we have tried the solution recommendations stated by the manufacture. When we ran a RT-PCR, we have been getting a small increase in fluorescence in the first few cycles, but then the fluorescence decreases. We have tried the default program setting (recommended by the manufacture) with similar results. I have run our samples with PCR to ensure that our primers work, which they do. With that being said any suggestions?

I am trying to get our RT-PCR machine up and running so that I can graduate sooner than later. With that being said, any help would be greatly appreciated.

We are trying to see any sort of amplification. Our reactions are 20 uL in capillaries with 2 uL of SYBR Green Master Mix, 2 uL DNA, different amounts of primers, and filling to a final volume with nuclease-free water. The PCR protocol we have is from the literature on very similar primers and we have tried the solution recommendations stated by the manufacture. When we ran a RT-PCR, we have been getting a small increase in fluorescence in the first few cycles, but then the fluorescence decreases. We have tried the default program setting (recommended by the manufacture) with similar results. I have run our samples with PCR to ensure that our primers work, which they do. With that being said any suggestions?

um...talk to me about your master mix?

many of those are supplied at 2X; do you use one supplied at 10X?

also, how are you setting up your RT reactions? and, what are the differences between the PCR you have done, and the RT-PCR you have attempted? is it only the master mix vs. buffer/taq/dNTP? or are there other components that differ?

what about controls? have you tried any sort of positive control?

Thanks for the response.

We ordered a startup kit (Roche) that came with 10X SYBR green master mix. We have been adding 2 uL of the master mix and our final volume is 20 uL. The PCR programs that we are using are from a paper similar to our project. Our PCR reactions have worked fine with our 2X master mix. The samples that we are running are DNA from ATCC.

I have been playing with the primer concentrations with the RT-PCR runs. The primer ranges have been from about 50 nm to 900 nm. I have been talking to some colleagues about this problem and they mentioned to try less concentrated primer concentrations.

We aren't concerned about the positive and negative controls yet because we are just trying to get the machine up and running. Our lightcycler has been here for about 2 years and the person trained in on it forgot his training.

Our PCR cycles are similar
PCR: 3 min at 95, Amp for 35 cycles of 1 min at 95, 1 min at 55 and 45 sec at 72, and final extension for 5 min at 72 (all in Celsius).
RT-PCR: 2 min at 50, 10 min at 95, Amp for 40 cycles of 45 sec at 95, 1 min at 55, and 45 sec at 72.

We have not played with addition MgCl2 yet. Our reference paper didn't use any so we haven't tried that yet.

I hope this information helps some.

I'm pretty new at RT also, but my first step would be to drop the annealing temp or run a gradient reaction. Some of my primers have been really sensitive to temp - as in product up to 58C, then nothing at any higher temp.

so the machine has been sitting there for a couple years, not being used?

hmmm....have you had a diagnostic run, to be sure it is in working order? it could be something silly like a bulb being burnt out

other question, if you take one of your RT-PCR samples afterward and run it on a gel, do you get a product? I am trying to determine if it is the machine or the PCR that is the problem

the only reason I would suggest a control...if your experiment is not working properly, often a control will tell you why, or at least which step to tweak. and I don't necessarily mean a control for your samples / setup / specific experiment; what about a control from your PCR--stuff vendor? something that should always work?

Thanks for the advice. I think that getting some positives is our next route to take.

Hello,

I was happed something similar with me and it was the machine (LightCycler 2.0). Roche bring me another one while they arrange them, so I can follow my experiments.
What happen to me was that the florescence decrease after some cycles, but if I run the samples after the PCR, I could see my aplimer. It was a problem with florescence detection.

A tip: I use the kit from Roche, and they advice to work with a final volum of 20 ul. It work as well with 10ul and you save SybrGreen, primer....

I wish that it may help you