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Why would identical PCR reactions work some times and not others?

Why would identical PCR reactions work some times and not others? - PCR - Polymerase Chain Reaction Forum

Why would identical PCR reactions work some times and not others? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 12-17-2010, 03:42 PM
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Default Why would identical PCR reactions work some times and not others?



I am doing large-scale PCR using two 384-well plates, run in the same thermocycler at the same time. All the wells have the same master mix distributed among them, including primers. The only difference between the wells is that DNA comes from different people. Yet, when I run the reaction, only about 10% of the wells show robust amplification, some have very little amplication, and majority none at all. What is the reason for such difference? Im pretty confident that DNA is not degraded, b/c I used the same batch to amplify a different gene, and it worked out fine. Moreover, when I repeat the reaction, different sets of wells amplify than before the results are not reprodusible. Im at a total loss please help!

Thank you,
Katya
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Old 12-17-2010, 06:32 PM
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Default Re: Why would identical PCR reactions work some times and not others?

I've done something similar in the past, although I have never done more than 70 reactions per run. I never had such an issue with them, I mean, from the 70, 50 or more did amplify, I can attribute my fails to the DNA (troublesome samples and stuff).

From my experience I can tell you that using thermal cyclers at their full capacity has detrimental effects on your results. In your case the problem is probably because the wells aren't heating uniformelly, and they do not reach the correct temperatures or get the correct incubation time with them.

Another issue is the size of your run, more than 700 rx! Adding the master mix plus the DNA ought to take you quite a bit of time, meaningwhile your mastermix reagents are degrading due temperature (even if your sitting them on ice), specially the polymerase. This could also be affecting your results.

Call me old fashioned, but I think pcr tubes (0.2 ml) are the best to make PCR rx's. They might take a bit more of time to prepare, but, to my taste, are more reliable than multi-well plates due to the uneven heating issues I mentioned above.

In conclusion, short and accurate is better than large and unreproducible. Hope I could be of help.

Last edited by luisillo; 12-17-2010 at 06:34 PM.
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Old 12-17-2010, 09:23 PM
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Default Re: Why would identical PCR reactions work some times and not others?

Thank you for your reply!

The thermocycler seems like a good theory, although I have run this reaction multiple times before with different genes, and those amplified well in the same cyclers. But it could simply be that they're getting old and not working properly, though my PI doesn't think that that is the problem, so I need to look for alternate explanations.

We have a robot mixer, so I definitely don't mix these hundreds of reactions manually - that would take all day! Which is why using tubes is infeasible, because the robot is designed to work with plates.

Thank you, again, for your feedback!
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Old 12-17-2010, 09:46 PM
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Default Re: Why would identical PCR reactions work some times and not others?

Yes, the cyclers don't get screwed that easily.

One question: Did you change the brand or the model of plates recently? Minimal misfit in the thermal cycler can cause that the wells don't receive heat properly, messing the reaction. Also, different plates might have different heat conductivity which might interfere with the incubations.

Another question: Did you already have this gene reaction (the one your stuck with) standarized? I mean, have you make it work properly? If not, It might just be that you need to standarize it further (i.e. adjust thermal cycles, reagent conc...), so you can get optimal results. I'm saying this giving that your DNA has worked with other genes.

By the way, If you are going to standarize, I recommend you make small size reactions (3-5, or the least that your robot mixer allows) in order to economize reagents.

Keep up the good work!
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