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How long can DNA precipitate at -20?


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6 replies to this topic

#1 jeanette

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Posted 09 August 2010 - 05:22 AM

Hi all,

I am extracting DNA from heavily lesioned grape leaves and as such, I have had to go to a really robust protocol in order to get any amplification in my pcrs because of all the polyphenolics and other fun contaminants. Of course, like many others, we are a 'poor lab' so using kits for extractions is out of the question. The problem is, this protocol takes forever (like 5 1/2 hours at warp speed) so high-throughput is difficult given my time constraints. It involves 2 precipitations....one with isopropanol and then one with sodium acetate and ethanol. I've let the precipitations go over a weekend with no discernible ill effects on yield or quality. So my question is, is there any papers anyone is aware of on extended precipitation times? I have searched and searched and have come up with nothing. And will my DNA degrade, like if I let it precip for a week? I'd like to break up this protocol into 3 stages so I am only working on extractions for like 2 hours out of my lab time....I work full time at another job so being able to split this up would be so much easier to increase throughput. Thanks for any comments.

jeanette

#2 Clare

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Posted 09 August 2010 - 05:58 AM

Others may say different, but I have kept DNA precipitating for months and it's fine (I always put mine at -80degC). In fact, I know a post-doc who actually stores her RNA in EtOH and sodium acetate (for long term storage in -80deg).
Clare

Hi all,

I am extracting DNA from heavily lesioned grape leaves and as such, I have had to go to a really robust protocol in order to get any amplification in my pcrs because of all the polyphenolics and other fun contaminants. Of course, like many others, we are a 'poor lab' so using kits for extractions is out of the question. The problem is, this protocol takes forever (like 5 1/2 hours at warp speed) so high-throughput is difficult given my time constraints. It involves 2 precipitations....one with isopropanol and then one with sodium acetate and ethanol. I've let the precipitations go over a weekend with no discernible ill effects on yield or quality. So my question is, is there any papers anyone is aware of on extended precipitation times? I have searched and searched and have come up with nothing. And will my DNA degrade, like if I let it precip for a week? I'd like to break up this protocol into 3 stages so I am only working on extractions for like 2 hours out of my lab time....I work full time at another job so being able to split this up would be so much easier to increase throughput. Thanks for any comments.

jeanette



#3 phage434

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Posted 09 August 2010 - 06:00 AM

No difficulty in letting the DNA precipitate in the freezer indefinitely.

I'd recommend a CTAB protocol for this application, if you are not already doing one.

#4 jeanette

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Posted 09 August 2010 - 06:19 PM

Thanks for the replies....I didn't think it would be an issue but wanted to hear others experiences. Yes I am using a CTAB protocol :-)

Thanks again,
jeanette

#5 hobglobin

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Posted 10 August 2010 - 10:26 AM

Thanks for the replies....I didn't think it would be an issue but wanted to hear others experiences. Yes I am using a CTAB protocol :-)

Thanks again,
jeanette

I wonder why you let the DNA precipitate this long...to my experience it won't increase the yield really...(or no cooling centrifuge available?). I only let the DNA precipitate if I've no time to continue working...

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

That is....if she posts at all.


#6 jeanette

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Posted 10 August 2010 - 04:17 PM

Hi Hobgoblin,
As I stated in my original post, it is only for my convenience. It's a long protocol and I have alot of samples to extract from...and I am not in lab full-time, as I have another job....so I got the idea of splitting up the protocol into 3 parts...so I can constantly be working on extractions but only taking a few hours out of my lab time after work to do them.

#7 bmb5yfl

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Posted 11 August 2010 - 07:40 AM

Hi,

I completely agree with Clare and phage434, precipitating DNA or nucleic acid in EtOH is perfectly safe. There is no aqueous condition for the enzyme to work so there is really no need to worry about degradation. The -80C is for long term storage as Clare stated and also to aid precipitation when the amount of nucleic acid is low.

I've actually left my DNA/RNA sample in 100% EtOH overnight on the bench before and the nucleic acids remained intact.




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