Using isopropanol in place of ethanol in DNA purification kits?

[jarlemag]

Hi,

The plasmid 'miniprep' extraction and PCR purification kits I've seen all require ethanol to be added to the column wash solution. Is it possible to add isopropanol in place of ethanol in these kits?

Best regards,

JP

[Bryan Jones]

I use "reagent alcohol" which is 90% ethanol, 5% isopropanol, 5% methanol, and it works fine. So you can at least replace some of the ethanol with isopropanol. I'm not sure what would happen if you just used isopropanol.

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[Cory Tobin]

I've used isopropanol for Qiagen minipreps before and it didn't make
any noticeable difference.

-cory

[Cathal (Phone)]

Isoprop is fine for washing minipreps. IIRC it has slightly different performance on RNA, and it will behave differently WRT salt also. But, for minipreps, it's not very important. You need a bit less of it than ethanol, it's a stronger precipitant.

Warning: DNA pellets washed with ethanol tend to become white and easily visible, whereas with isoprop they can be glassy and transparent.

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[Nathan McCorkle]

From the mini-prep folks:
https://www.qiagen.com/us/resources/molecular-biology-methods/dna/#Isopropanol
precipitation of DNA

Mentions better precip at warmer temps, better salt solubility (which
leads to harder to see glassy pellets rather than clouded-with-salt
pellets)... but the main point I remember: downstream processes could
be affected -- because isopropanol is less volatile it dries more
slowly or not completely if you are too fast... slower drying gives
more time for energy-state minimization, thus when it is dry it is
harder to redissolve (it must be denser and more closely-packed I
guess).

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[jarlemag]

Thanks for the advice, everyone. I'm familiar with the differences in pellet visibility, but I didn't know about the difference in salt solubility. In any case, I'm not concerned with manual pelletting and washing, but spin column kits like this one: http://dongshengbio.com/en/xxcp.asp?id=271

Best regards,

JP

[Nathan McCorkle]

On Sat, Jul 23, 2016 at 1:46 AM, jarlemag <jarle...@gmail.com> wrote:
> Thanks for the advice, everyone. I'm familiar with the differences in pellet
> visibility, but I didn't know about the difference in salt solubility. In
> any case, I'm not concerned with manual pelletting and washing, but spin
> column kits like this one: http://dongshengbio.com/en/xxcp.asp?id=271

As described in that protocol "Discard the flow-through", the reason
for the alcohol is to remove salts or other impurities. Because
ion-affinity chromatography is based on charge, this is largely based
on pH, but salts also affect this and sometimes the buffers include
salts specifically to enhance binding for example. So use less
isopropanol in place of the ethanol.

P.S. Cory confirmed he has tried this with spin-columns and it works

[Cory Tobin]

P.S. Cory confirmed he has tried this with spin-columns and it works

Yup, I've used isopropanol with the Qiaprep Miniprep kit.  Worked just fine and gave me ~300ng/uL plasmid.

-cory