Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

gDNA purification - anything better than alcohol precipitation? (Jan/29/2009 )

Pages: Previous 1 2 

If you saw the DNA bands pre- and post-ethanol precipitation were indistinguishable in the gel, then there was no DNA loss. All you "lost" in ethanol precipitation was the mono-, di-, and other oligonucleotides from RNA degradation by the RNase treatment. How did you extract the DNA and have you sent it for sequencing yet? Send it out and you'll know the result in a couple days.

-AquaPlasmid-

AquaPlasmid on Jan 30 2009, 06:33 PM said:

If you saw the DNA bands pre- and post-ethanol precipitation were indistinguishable in the gel, then there was no DNA loss. All you "lost" in ethanol precipitation was the mono-, di-, and other oligonucleotides from RNA degradation by the RNase treatment. How did you extract the DNA and have you sent it for sequencing yet? Send it out and you'll know the result in a couple days.


well, I don't seem to be able to explain myself, sorry. The bands were the same in their positions. This means, to my knowledge, that the composition of whatever nucleotides in both samples was the same. The intensity was only slightly weaker in the pre-precipitation. Post-precipitation the concentration should have been 10 times higher (as the volume went from 200 to 20 L), and thus the bands should have been much more intense. If the intensity would have been EXACTLY THE SAME in both samples, then that would have meant that I lost not half but around 90% of the DNA!

-ditaviz-

ditaviz on Jan 30 2009, 11:12 AM said:

AquaPlasmid on Jan 30 2009, 06:33 PM said:

If you saw the DNA bands pre- and post-ethanol precipitation were indistinguishable in the gel, then there was no DNA loss. All you "lost" in ethanol precipitation was the mono-, di-, and other oligonucleotides from RNA degradation by the RNase treatment. How did you extract the DNA and have you sent it for sequencing yet? Send it out and you'll know the result in a couple days.


well, I don't seem to be able to explain myself, sorry. The bands were the same in their positions. This means, to my knowledge, that the composition of whatever nucleotides in both samples was the same. The intensity was only slightly weaker in the pre-precipitation. Post-precipitation the concentration should have been 10 times higher (as the volume went from 200 to 20 L), and thus the bands should have been much more intense. If the intensity would have been EXACTLY THE SAME in both samples, then that would have meant that I lost not half but around 90% of the DNA!


Aha... we all mis-communicated here. Maybe you should run eq. amount (e.g., take 2ul post- and add 80ul water, then load 10ul to gel for both pre- and post-). I meant you might have degraded RNase products in your pre-ethanol sample, which are tiny and give strong A260 reading and may overestimate your DNA concentration. After you ethanol the gDNA, these free nucleotides and oligos from RNase treatment, and even small RNAs, would be gone and give you the apparence of DNA loss.

-AquaPlasmid-

Evaporation in H2O increases recovery in concentration.

I'm still waiting for the glycogen, meanwhile extracted and eluted (same sample) in water or buffer AE (QIAGEN), then evaporated the water elutions in dessicator, after 10 min centrifuging at full speed (16.4 K G), and precipitated the AE elutions with Alcohol-Na-Ac protocol as usual.

For samples with at least 3g of DNA I get between 2% and 20% loss with evaporation (where the h does it go??? I rinsed the tube as much as I could, and centrifuged several times during the evaporation), and around 30% loss with precipitation.

For samples with less than 3g DNA I got between 16% and 30% loss with evaporation, and around 50% loss with precipitation.

Pre-concentration there was practically no difference betwen eluting with AE buffer and water. The bands look also quite similar, and both elutions give quite clean DNA, judging from the gel and from the NanoDrop values.


So, water elution-evaporation is winning. Still I would like to lose rather 2% and not 20%. Next week I'll try with the glycogen, and with supernatant recovery and re-precipitation.

Thank you all for the comments and ideas!

-ditaviz-
Pages: Previous 1 2