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Aaaah I want to die!!!! PCR won't work - Why do the extractions that amplified 2 weeks ago fail now? (Apr/12/2010 )

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Hi All

Been having problems with my PCR. Trying to amplify DNA from bat fecal samples. All was fine at first, using Qiagen DNEasy kit -- then I learned the hard way that these extractions were not stable at 4C for more than a month. I've had to do a bunch of re-extractions, using various kits (including Qiagen stool kit). Once I confirmed that I was getting PCR product, I archived half the sample immediately at -20C, then kept the working aliquot the fridge for <1 week while I ran some additional PCR's, then moved to freezer.

Well, now I want to redo some of these PCR's (cloning didn't work out so good the first time, need to start over). But now I can't get these flipping samples to amplify!!! Same primers, taq, dntp's, buffer... but nothing!!! Every once in a blue moon, I get one band from maybe one out of 15 samples -- but my postitive control, extracted less than a month ago -- NOTHING!

So I went back to the "archived" samples -- maybe being in the fridge for that little while screwed them up? Same damn thing. Three out of 20 amplify, rest don't.

I am completely flummoxed. Can my extractions really have degraded THAT QUICKLY??? I would say that the taq or dntp's had gone off, but then I do get a one or two to amplify with every batch I run. I am going to try again today with totally new taq, buffer, and dntp's -- but does having a couple amplifications per run negate this? Is there anything I can do to make these extractions work again?? I don't understand how they could have degraded at -20C.

I am very sad... ;)

-KimWG-

Freeze - thawing samples like DNA is not good for them. If they were already partially degraded, a single freeze/thaw step might be enough to push them over the edge, as it were. What solution are you storing your DNA in (water, tris, TE)? Also, check the temperature of the freezer, it may not be freezing your samples, so they remain liquid and get degraded by DNases in solution.

I would first test new dNTPs and primers, these are the PCR components that degrade most commonly, before suspecting the samples though.

-bob1-

bob1 on Apr 12 2010, 07:24 PM said:

Freeze - thawing samples like DNA is not good for them. If they were already partially degraded, a single freeze/thaw step might be enough to push them over the edge, as it were. What solution are you storing your DNA in (water, tris, TE)? Also, check the temperature of the freezer, it may not be freezing your samples, so they remain liquid and get degraded by DNases in solution.

I would first test new dNTPs and primers, these are the PCR components that degrade most commonly, before suspecting the samples though.


An interesting consideration... samples are stored in TE buffer, but they did degrade after <3 months at 4C. I think the freezer is OK, otherwise there would be a lab full of very sad people; and also, everything is frozen when I take it out. I'm going to see if I have a different primer set, too -- that could be an issue, and one I haven't tried yet. Thanks.

-KimWG-

Have you re-measured your DNA after thawing? Make sure your DNA solutions are homogenized after thawing. DNA tend to stick together during freezin and if you thaw and use 1Ál directly to your PCR without any easy vortexing or shaking you might very well get no DNA to your reaction. I've always heard that vortexing genomic DNA is a no-no because of thread disrutance but for most application other than library cloning vortexing works just fine for me. We got much better results after we introduced a vortexing step in our protocols (genotyping, SNPs etc)

-DocFlow-

Hi I had a similar problem and I found that titration of template helps. Try to dilute your template 1X 5X 10X 50X 100X or even more 500X, 1000X etc. You will probably find the dilution which will work for all other samples. I think it is caused by inhibitors in sample, escpecially when isolated from feces.

-vladooo-

AND: DNA does NOT degrade when frozen (almost). Look at as they found mammoth DNA 37 000 years old. Your life is too short to detect any degradation of frozen sample. :lol:

-vladooo-

vladooo on Apr 14 2010, 03:44 AM said:

AND: DNA does NOT degrade when frozen (almost). Look at as they found mammoth DNA 37 000 years old. Your life is too short to detect any degradation of frozen sample. :(


I was thinking almost that same thing! They just isolated mtDNA from a human 40,000 year old human finger bone found in Siberia, and were quite surprised to find that it was neither neanderthal nor modern human, but from some third lineage that had diverged from ours approximately 1 million years ago. I was reading that, wondering how if they can pull that off, why the hell can't I get a 500 bp segment of mitochondrial DNA from bat turds that have been in the freezer for, like, 2 years.

I suspect that dilution is not the answer to my prayers here (these very same extractions were working at "full" strength just a few weeks ago), but I will give in another shot (at stronger dilutions) just to make sure...

-KimWG-

DocFlow on Apr 14 2010, 12:30 AM said:

Have you re-measured your DNA after thawing? Make sure your DNA solutions are homogenized after thawing. DNA tend to stick together during freezin and if you thaw and use 1Ál directly to your PCR without any easy vortexing or shaking you might very well get no DNA to your reaction. I've always heard that vortexing genomic DNA is a no-no because of thread disrutance but for most application other than library cloning vortexing works just fine for me. We got much better results after we introduced a vortexing step in our protocols (genotyping, SNPs etc)


INTERESTING. I spun em down but did NOT vortex first because of the multiple warnings of terribly dire consequences for all. Of course, I'm trying to amplify a 500-bp segment of mtDNA so that might not matter, but "better safe than sorry" was what I was thinking. I will definitely try this as they can't get any more not working, right?

-KimWG-

KimWG on Apr 14 2010, 12:30 PM said:

DocFlow on Apr 14 2010, 12:30 AM said:

Have you re-measured your DNA after thawing? Make sure your DNA solutions are homogenized after thawing. DNA tend to stick together during freezin and if you thaw and use 1Ál directly to your PCR without any easy vortexing or shaking you might very well get no DNA to your reaction. I've always heard that vortexing genomic DNA is a no-no because of thread disrutance but for most application other than library cloning vortexing works just fine for me. We got much better results after we introduced a vortexing step in our protocols (genotyping, SNPs etc)


INTERESTING. I spun em down but did NOT vortex first because of the multiple warnings of terribly dire consequences for all. Of course, I'm trying to amplify a 500-bp segment of mtDNA so that might not matter, but "better safe than sorry" was what I was thinking. I will definitely try this as they can't get any more not working, right?

I work with plasmid samples of around 5kb. And I always vortex my templates after I thaw them. Never had a significant problem from vortexing. So I think your 500-bp DNA fragment should be fine.

-ProteinWork-

vladooo on Apr 14 2010, 03:44 AM said:

AND: DNA does NOT degrade when frozen (almost). Look at as they found mammoth DNA 37 000 years old. Your life is too short to detect any degradation of frozen sample. :(

Half right there, true it won't degrade if frozen solid indefinitely, but thawing it out will degrade it quite a bit. Also, some DNases are partially active at -20 (or so it seems) as samples are known to be degrading at these temperatures. -70 works though.

KimWG on Apr 14 2010, 08:27 AM said:

I was thinking almost that same thing! They just isolated mtDNA from a human 40,000 year old human finger bone found in Siberia, and were quite surprised to find that it was neither neanderthal nor modern human, but from some third lineage that had diverged from ours approximately 1 million years ago. I was reading that, wondering how if they can pull that off, why the hell can't I get a 500 bp segment of mitochondrial DNA from bat turds that have been in the freezer for, like, 2 years.


KimWG on Apr 14 2010, 08:27 AM said:

bat turds

I think that's the important part here! Faeces are full of bacteria (which excrete DNases) and all sorts of chemical compounds like acids and bases, excreted compounds, degraded cell compounds, food residues (if the bats are herbivorous many plant compounds inhibit PCR extremely strongly) etc. This is more or less equivalent to leaving a hunk of meat sitting outside in a tropical climate for a month and then trying to extract DNA from it for PCR.

-bob1-
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