6 replies to this topic

[Alexandre]

Hi,
I'm having a problem with my real time PCR reactions using molecular beacons. Some fluorescence curves exhibit irregularities different from the standard curve. It will degradation of the molecular beacon or primers? See the picture below. Can anyone help me?



Thank you.

Edited by Alexandre, 19 August 2012 - 06:44 AM.

[Curtis]

All your samples have the same problem?

[ascacioc]

To me, on a first impression, it looks like you have to little template in the reaction and that left longer the PCR would actually reach the plateau.

Andreea

[Alexandre]

This problem occurs in some reactions isolated, but not in all reactions. This pattern also occurs in the negative control. It would be some interference of primers or samples?

[ascacioc]

If it does that in the negative control, I would put my money on the primers being the problem. Check them for secondary structures, homodimer or whatever else they could do that can interfere...

Andreea

[Trof]

Are those problematic reactions on the same run as the OK ones? Do the problematic reactions have some kind of pattern, same wells/wells on the edge?

I've seen such jagged baselines on plates run multiple times (unused wells) on LC480. We are not sure of the reason, one may be the increased elasticity on a used plate and increased evaporation for that reason.
You seem to have ABI Prism however, so you maybe don't even use plates.

[ascacioc]

Now that you mention this pattern, it reminded me of an article I was reading once about different qPCR machines and comparison between heating on the different spots on the plate: apparently some people test it (I don't have the article anymore even though I should have kept it for future reference) and they discovered that the different spots on the plate of different cyclers are heated differently (they even offered maps for different companies) and this can contribute a lot to your experiment because you have differences depending on where you put your sample. Bottom line: check the pattern of where the tubes are situated. Do the negative/positive control with different positioning in the machine. It is only one run: 5 tubes with + control and 5 tubes with - control and see what happens.

Andreea