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PCR trouble

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PCR trouble

Postby Harmakhis » Wed Jul 06, 2011 11:20 am

Hello,

I have a rather odd problem with my PCR... I'm performing microsatellite analysis of molted feathers. The DNA isolation is done with a standard tissue kit and after that the PCR. For the past six months everything was fine and then suddenly after one weekend nothing works anymore.
The PCR products are used for genotyping (with MegaBACE) and for the past two weeks I don't get any results. I tried visualizing the product via EtBr gel: nothing as well. The funny thing is that I didn't change anything.

Anyway, here is what I tried: I used new polymerase, new dNTPs, new buffer and new water. I exchanged the primers and tried the PCRs on different thermocyclers, I check the reaction programs as well.
I used Nanodrop to be sure I have template DNA: It's around 10 ng/µL in each sample, which seems a bit low but considering I used feathers as a source I guess that's fine, especially as it worked all the months before.
I tested the PCR products with Nanodrop as well and got a consistent 210 ng/µL which seems it quite low. So there really seems to be a problem with the reaction.

But I'm at a loss here. Any suggestions would be highly appreciated.
Harmakhis
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Postby canalon » Thu Jul 07, 2011 4:08 pm

Do you have a positive control (DNA that previously amplified, or better plenty of highly purified plasmid from another source) that you can test. Because the problem might come from your DNA extraction.
Did the problem started with you changing one of the reagents. For example new dNTPs. It has happened to me that a series of working solutions were prepared at the wrong dilutions and were poisonning the PCR, taking a new one did not improve things, because they were also too concentrated...
A good check is to test your PCR reagents on a completely unrelated and easy PCR. Like direct colony PCT of 16S DNA or whatever very basic, common, no fail, PCR you usually do in your lab.
And to make sure it is not a staining problem, you see the molecular weight marker on your gel, besides the empty lanes, yes?
Patrick

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Re: PCR trouble

Postby Harmakhis » Fri Jul 08, 2011 9:33 am

Yes, I do have a positive control. But it doesn't work as well. I already exchanged all stock solutions and tried again, until now with no luck. I thought about the staining as well, but I can see the ladder and the problem persists when using MegaBACE for genotyping. So I guess it's not the gel.

Unfortunately I can't try an "easy" PCR as we don't have stuff like 16S DNA available, we only work with extracted DNA from vertebrate for population genetic studies. But I will try some completely unrelated samples and primers.
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Postby canalon » Fri Jul 08, 2011 3:22 pm

So you know it is something in your PCR reaction that is start.
I would next run 3 PCR:
- your old control with a different set of primer (another gene)
- new sample with regular primers
- new sample with the same set of primer as number one.
That should sort out if your problem is with primers or PCR reagents.
Then if all those fail, find someone who has a PCR that works and try to replace their reagents with each of yours in turn (or at the same time in different tubes).

If all else fails sacrifice an undergrad to the PCR gods at the next rising full moon (i.e. accuse them falsely of the worst you can think of, and that they cannot understand and send him/her home confused and lost). Swear loudly and prepare the next PCR fully drunk. That probably won't help, but at least you will have an excuse for this failure. And might even had a good time drinking.
Patrick

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