I wonder if anyone has a positive experience for improving DNA quality following extraction with Chelex? This method is great for improving the yield, particularly for samples low in DNA or material, but it is also known for resulting in low quality DNA. The absorbance spectra are generally poor. Some methods employ proteinase treatment in concert with Chelex, but in our hands this does not improve the absorbance spectra in a remarkable way. Therefore, I would appreciate any suggestions on how to improve quality of DNA extracted with Chelex.
Hi,I am not sure what do you mean by "The absorbance spectra are generally poor". Boling with Chelex solution release DNA because you break the cells, inhibits DNases and and at the same time protects DNA form boling! So you end up with DNA in solution but you may have a bit of everything not a pure nucleic acid preparation.
For PCR Cheelkx-Dan is fine, for otehr things: I treated with Prot. K and then cleaned Chelex-DNA with standar DNA columns or Phenol/Ethanol purification. This clean de DNA for downstream molecular biology applications..i..t but reduces DNA concentration.
Thanks, Pedro. Chelex basically inactivates enzymes by absorbing metal ions (mostly Mg++) and increasing pH, but does not do much more than that. What you call "bit of everything" is a lot of everything:)
After cooling down, the DNA restores its ds structure and should have a clean peak at 260 nm. Problem is that it does not show a sensible peak - at least, for our samples and quantities of impurities. PCR does work, this is true, but metagenomics - not always. Columns seem to deplete the amount of the DNA unacceptably (this is what we saw in the first trials anyway)... I am perhaps looking for a magic bullet...
Hi. we often use of DNA purification by Phenol-Chloroform and column purification. these methods are so impressive.
Hi Ehsan, yes, they are, but not for small samples - too much losses.
I guess the problem is not the chelex, it is the agresive breakdown of the cells. Did you try to precepitate with chilled Ethanol? or chilled ethanol+Na-acetate (for better recovery)?
Hmmm... what do you mean by aggressive breakdown? The tissues are not smashed in a FastPrep or anything like that and the DNA is not fragmented...
We don't precipitate with EtOH, not enough material for that:(
Elena-- did you ever have any luck with purifying your DNA samples post Chelex extraction? I'm interested in an update if you have one. Thanks!
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