Cesium-cleaned environmental DNA - Help! - Running into problems after extracting ethidium (Jun/11/2008 )
Hi All,
We routionely extract DNA from environmental samples (dirt, mud, etc). The DNA is genomic DNA, from any microorganisms that may be present. This DNA often needs to be 'cleaned' from 'inhibitory substances' (various chemicals in mud that co-purify with DNA) by running the DNA through an EtBr-cesium gradient.
So far so good.
After the gradient, the genomic DNA appears as a band which is pulled, and the Ethidium is extracted from the DNA with Isoamyl alcohol or Butanol. The DNA is then concentrated in a centricon (Amicon) to remove salt and reduce volume.
Here's the problem. The Centricons are not supposed to be used in the presence of these organics (trace butanol etc in the DNA, from the previous step). The trace butanol seems to compromise the membrane, and the DNA is not retained - it flows through the membrane with everything else.
So here's my question. What happens if I run the cesium gradient without ethidium? Will the DNA (which is linear genomic DNA) still band (I realise I won't be able to visualise it).? If I can do the gradient without ethidium, I can avoid the butanol and go straight to the centricon, and it should retain the DNA.
Alternatively, are there other *easy* ways of removing ethidium? I don't know how to use Dowex and don't have time to develop ion-exchange, which is what Maniatis recommends as an alternative. Anyone have any other ideas?
Can ethidium be extracted with phenol? Phenol seems to be OK to use with the columns.
Thanks for any tips.
Patty
Patty,
First, DNA will have a different density without EtBr, so I wouldn't omit it from your spin.
I would think that removing the CsCl would be much more important than removing EtBr (for later PCR rxns anyway). I used to prepare plasmids by a similar method as yours (through the n-butanol step) before Qiagen made life easier. I think the step after Butanol was ethanol precipitation (probably with sodium acetate). This got rid of the cesium and allowed the resuspension to the desired DNA concentration.
You might check Qiagen or Stratagene for quick ways to purify genomic DNA that don't require CsCl or EtBr.
-dan
First, DNA will have a different density without EtBr, so I wouldn't omit it from your spin.
I would think that removing the CsCl would be much more important than removing EtBr (for later PCR rxns anyway). I used to prepare plasmids by a similar method as yours (through the n-butanol step) before Qiagen made life easier. I think the step after Butanol was ethanol precipitation (probably with sodium acetate). This got rid of the cesium and allowed the resuspension to the desired DNA concentration.
You might check Qiagen or Stratagene for quick ways to purify genomic DNA that don't require CsCl or EtBr.
-dan
Thanks Rosewater - Plasmids can be precipitated but in my understanding this approach is dicey with genomic DNA. So, centricons.
I've been through the kits and haven't found anything that will work well enough, sadly. I am thinking that phenol or chloroform should remove butanol from the aqueous phase, but am not sure..... ??
I suppose I could dialyse the ethidium away, but we start with so little (~5 micrograms) I hate to add steps that could carry a loss.
We routionely extract DNA from environmental samples (dirt, mud, etc). The DNA is genomic DNA, from any microorganisms that may be present. This DNA often needs to be 'cleaned' from 'inhibitory substances' (various chemicals in mud that co-purify with DNA) by running the DNA through an EtBr-cesium gradient.
So far so good.
After the gradient, the genomic DNA appears as a band which is pulled, and the Ethidium is extracted from the DNA with Isoamyl alcohol or Butanol. The DNA is then concentrated in a centricon (Amicon) to remove salt and reduce volume.
Here's the problem. The Centricons are not supposed to be used in the presence of these organics (trace butanol etc in the DNA, from the previous step). The trace butanol seems to compromise the membrane, and the DNA is not retained - it flows through the membrane with everything else.
So here's my question. What happens if I run the cesium gradient without ethidium? Will the DNA (which is linear genomic DNA) still band (I realise I won't be able to visualise it).? If I can do the gradient without ethidium, I can avoid the butanol and go straight to the centricon, and it should retain the DNA.
Alternatively, are there other *easy* ways of removing ethidium? I don't know how to use Dowex and don't have time to develop ion-exchange, which is what Maniatis recommends as an alternative. Anyone have any other ideas?
Can ethidium be extracted with phenol? Phenol seems to be OK to use with the columns.
Thanks for any tips.
Patty
Hi Patty,
I used to prepare plasmid DNA using the CsCl preps. In order to remove the EtBr we used isobutanol and then we dialysed v TE over a 16-24hr period at 4oC with several changes to get rid of the isobutanol. This worked fine for us. auldmok
Why not use the centricons to concentrate and remove CsCl first, and then worry about the ethidium bromide?
Oooh, lots of ethidium waste, - I dunno, maybe.
What I'm trying today is an overnight spin without ethidium. It's not a banding, but a precipitation and hopefully the DNA will pellet on the bottom away from the inhibitors - which tend to be less dense. Anyway, I'll ressupend the pellet and try the centricons on that tomorrow.
Is it plausible that with low starting amounts of DNA (ie only a few micrograms) that it all binds to the membrane?
Also, are there dialysis filters (discs) that I could try, that would let me avoid tubing (which is sure to carry a loss)? ANyone have any experience with dialysis filters?
Alrigh another Q:
Can butanol 'go bad?' I am starting to think that my problem may not be the filters but the organics. I know that phenol can go off and be very damaging to DNA as a result. Can other organics, like butanol, do the same thing?