Cleaning up dirty gDNA (desperate...) - (Nov/11/2008 )

Hi,

I'm struggling with something and I could realy do with advice...

I am trying to genotype 1000 voles, and have the DNA extracted already. The problem is the DNA was extracted before I arrived, crudely, by alkaline digestion, which basically involved placing a blood sample in a tube with ammonia solution, heating to 100 degrees C and then evaporating off half of it... As you can imagine the soltuion is a murky brown colour, doesn't look nice.

Now, this DNA PCRs well, so it's definitely in there. The problem is it won't work for genotyping assays, maybe because of pH, maybe because of the other crap in there, I don't know. I'm desperately trying to clean up my DNA (it's the basis of my whole PhD) but don't know how to - all commercial gDNA clean-up kits are for starting with tissue, or blood etc. I tried TriZOL but no luck. Can anyone think of a way to clean up what I have - no cells, just already-extracted genomic DNA - to leave me with just the gDNA and no horrible stuff?

Thanks, I hope I haven't waffled and that this makes sense.

Andy.

-andykt-

It appears to me that I have met this sort of problems before.

How about taking a Nested PCR by using your crude DNA PCR in the first place?

Or using ethanol or isopronoal precipitation of your genomic DNA?

-Wolfgang-

Hi Andy,

Why don't you just precipitate the DNA? That's what I do when my gDNA samples are "dirty".

Add 2 vol EtOH
1/10th final vol NaOAc (3M stock)
Spin 20 mins, 4deg, max speed (ie: 14K in a benchtop centrifuge).
Remove SN
Wash with 70% EtOH
Spin again (10 mins)
Remove SN
Air dry pellet, resuspend in water or buffer

Easy I mean, you'll have to do it 1000 times for all your samples, but at least it's do-able!

Clare

-Clare-

Clare,

I had a feeling there'd be something easy I could do...! So thanks very much for that idea, I'll give it a shot!

Andy.

QUOTE (Clare @ Nov 11 2008, 04:26 PM)

I mean, you'll have to do it 1000 times for all your samples, but at least it's do-able!

Clare

-andykt-

I agree with Clare: Ethanol Precip always works for me - I do extractions from fecal samples that are always so dark at completiton.

-Jae-

Try the suggested precipitations on a few samples and see how it works. You may have to go with phenol/chloroform extractions, and then ethanol precipitation. But with 1000 samples, good luck (if you have to do the P/C extractions, use a fume hood!). I don't see why any standard DNA extraction kits wouldn't work, though.

-wbla3335-

Hi Andy,

No worries How did you get on?

Clare

[quote name='andykt' date='Nov 13 2008, 02:43 PM' post='157240']
Clare,

I had a feeling there'd be something easy I could do...! So thanks very much for that idea, I'll give it a shot!

Andy.

-Clare-