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Isopropanol precipitation - best time and temperature?


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#1 rybkasusa

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Posted 02 December 2004 - 07:43 AM

Hi,

does anyone know what is the best temperature and time for Isoprop precipitation of DNA? I usually do at room temperature and centrifuge immediately. Can I improve this? And would it matter if I keep my samples on ice for 1-2 hours before centrifuging?
I am courious to hear your opinion about this!

Rybkasusa

#2 Simonsays

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Posted 02 December 2004 - 12:04 PM

I precipitate DNA with isopropanol, 10 minutes, on the bench. I then centrifuge, discard the supernatant, wash with 70% ethanol (-20C), centrifuge, discard the supernatant, and dissolve my DNA pellet in water about 60 minutes at 37C.

Hope this helped

Simon

[I]I don't think 1-2 hours on ice would change much... but I might be wrong...

Edited by Simonsays, 02 December 2004 - 12:05 PM.


#3 Solomona

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Posted 03 December 2004 - 01:14 AM

I would say this depends on the salt concentration in precipitation. When you do not add NaAc you should definitely put the vial on ice for some time and centrifuge at 4 C.

I let most precipitations stand at 4C over night or incubate them >30 min. on ice and never add NaAc, because it can co-precipitate salts.

#4 rybkasusa

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Posted 03 December 2004 - 06:00 AM

So you precipitate without salt? How does this work?

#5 Solomona

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Posted 03 December 2004 - 06:35 AM

There are salts inside your extract, like Tris or NaCl.
Adding 0,1 V 3 M NaAc is increasing the precipitation of nucleic acids, as well the precipitation of other salts.

Precipitating 30 min. on ice without adding anything besides Isopropanol gives same yield like 30 min. on ice and 0,1 Vol. NaAc. It is just decreasing the time. Add it when you have little DNA in your sample and definitely leave it away when you know there is a high salt background matrix in your extract.

Recently I compared temperature, incubation time and NaAc addition because I had a contamination carried over from extraction and found that precipitating in room temperature gives much smaller yield than on ice and NaAc is just shorting the precipitation time. That screening didn't really help with the co-precipitation of humic acids but made me feeling good about the standard precipitation in our lab.

So, when you are not in a hurry, do you extractions in the afternoon, put you samples on ice or in the fridge and be happy with it next morning. One hour will be enough in most cases.

regards,
Solomona

#6 rybkasusa

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Posted 06 December 2004 - 03:51 AM

Now I finally tried with Isoprop (and o,3M Sodiumacetate), keeping on ice for 2 hours (because we had a seminar then B) ), and had really nice amounts of DNA.




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