4 replies to this topic

[science noob]

Was using the Qiagen Midiprep kit + Qiafilters (after adding solution 3 of the plasmid prep) - everything was fine (floating lysates etc).

At the precipitation step, I added absolute ethanol instead of isopropanol to my DNA elute and didn't even see a trace of pellet (which I was expecting to see) after centrifugation (spun at 15,000g 30min 4C).

Will the use of ethanol instead of isopropanol affect DNA precipitation??

Just another note, the Qiagen kit was dated 2009 - solution 1 and 3 were appropriately stored at 4C.

Edited by science noob, 03 July 2012 - 08:27 PM.

[aehrens]

Yes, it will affect the efficiency of precipitation. When using isopropanol, it is enough to add 1 volume per volume dna, whereas for ethanol you have to add at least 2.5 volumes for the dna to precipitate.

[Curtis]

Why do you have to centrifuge for 30min?

To add to aehren's comment, I have to admit that I have accidentally added 70% ethanol instead of iso-p many times, but I still managed to pellet down my plasmids. However, I normally top up the tube with alcohol. Therefore, technically it's more than 1 volume per volume DNA.

[science noob]

Curtis, on 03 July 2012 - 09:48 PM, said:

Why do you have to centrifuge for 30min?

To add to aehren's comment, I have to admit that I have accidentally added 70% ethanol instead of iso-p many times, but I still managed to pellet down my plasmids. However, I normally top up the tube with alcohol. Therefore, technically it's more than 1 volume per volume DNA.

15,000g 30min 4C was according to the Qiagen Midiprep protocol after addition of isopropanol.  My guess it that it will pellet the DNA?

[Nabin]

I had similar problem with my maxi prep when I did it for the first time, i figured it out later that I forgot to mix isopropanol with the eluted solution, I just added isopropanol ,kept it at -20 C overnight and centrifuged the other day. I hope you didn't do that.  The issue is the liquids do not mix each other properly so when you just add alcohol to them, it just stays on the top and it is unable to precipitate the DNA from the eluted product.