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Cleaning up old (not quite ancient) gDNA extractions


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#1 Stapler12

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Posted 25 March 2011 - 08:51 AM

Hi,

I've just started on a project that was started quite a few years ago and am having a bit of trouble. I have inherited a few hundred gDNA extractions (of birds) that were done a number of years ago (probably more than 10) which I would really like to be able to use as they have excellent field data associated with them.

However, the extractions seem pretty poor. Despite often having good concentration readings on the nanodop (20-120ng/ul), their ratios are rather worrying, typically 260/280 readings are between 1-1.5 and 260/230 are all less than 1. If I run these samples out on a gel, most do not show a blob of DNA.

So far I have tried clenaing them up with a QiaAmp micro kit, but this just seems to lower the apparent DNA concentrations and has no real effect on the 260/280 or 260/230 ratios.

I am afraid I have no idea (and no-one can remember) how these samples were prepared or even what tissue they were from. Although some of the tubes look a bit furry, which is odd as they are birds!

Do you think there is any hope for these samples? Could the "DNA concentration" be purely contaminants (seeing as I am not getting any bands on a gel).

Any tips on how I might salvage these samples would be greatly appreciated!

With many thanks

#2 Ameya P

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Posted 25 March 2011 - 10:11 PM

Stapler,

Could you tell us what are you planning to do with the DNA you get? That would help making suggestions.

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#3 pDNA

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Posted 25 March 2011 - 10:59 PM

if the agarose gel says no DNA ...then there is in most cases no DNA!
What do you use for staining of your gels?

Regards,
p

#4 Stapler12

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Posted 28 March 2011 - 12:40 AM

Hi both,

Thanks very much for your replies.

I was hoping to use the samples to SNP genotype the birds. I believe I would need 250ng of DNA at a concentration of at least 50ng/ul to do this. This is probably quite unrealisitic for the quality of the samples, but being able to pedigree them and PCR a couple of genes would certainly be useful.

I am running out the samples using Web Green dye, we don't use ethidium in our lab as we have a girl who is pregnant.

Thanks for any more advice!

#5 hobglobin

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Posted 28 March 2011 - 01:07 AM

Of course DNA can now be quite degraded after this years depending on storage conditions and preparation. But an empty gel can also indicate that the DNA concentration is just too low to be visible (no idea about the detection limits of Web Green dye). But anyway a PCR still might work with this low concentration...i.e. I'd try out a PCR with a primer from which you know that it works (and that you later can use as positive control).
DNA isolation with standard kits might be not useful, because the loss can be quite high (though this kit is developed for this type of samples).

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#6 phage434

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Posted 28 March 2011 - 12:51 PM

I would not dream of trying to see a valuable, ancient DNA sample on a gel. If you have good primers, you should be able to amplify a sample with little trouble. See the enzyme mixes available from e.g. NEB to repair damaged DNA. A gel is just wasting your DNA sample, and is unlikely to reveal anything of interest.




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