Methods for desalting - (Jul/14/2010 )

Hi everyone,

The extraction buffer I use for extracting DNA from bones has 0.5M EDTA and even after purifications steps (phenols and/or Qiagen MinElute), I suspect I still have quite a lot of salt.
Do you know an easy way to desalt BESIDE ethanol precipitation? (I don't like precipitating because I'm worried to lose the pellet).
Also, is there a way to quantify the amount of salt that is still in my extract?

Thank you

Maddie

Dialysis, ultrafiltration, solid phase extration.

I think she is looking for a simple protocol she can use without any help of machines?

I do wonder why you do not want to use the ethanolprecipitation? I have had good results with it, however you do seem to lose some DNA.
Have you tried it before or?

and what about a kit (gelextraction kit or so?)

Those methods do not require any specialized equipment, just materials:
Dialysis = refrigerator, magnetic stirrer + dialysis tubing
Ultrafiltration = centrifuge + ultrafiltration tube
Solid phase extraction = syringe + SPE column

With specialised equipment, I was allready thinking of dialysis tubing, ultrafiltration tubes and SPE column..
I know that in a lot of labs you will have one or more of those, but at the lab were I worked, we didnt have any of them:p nor the money to buy any of them haha.

No I don't have dialysis tubes etc..
I did ethanol precipitaions yeaaaars ago when I was cloning, but I had lots of DNA that I could easily see the pellets.
Now I want to clean an extract of ancient DNA, thus small fragments and small quantities. I guess I won't see much of a pellet and I could lose the whole thing.

What are the ultrafiltration tubes? I don't have any but could maybe ask other dept that are around? What would be the safest way for my kind of sample out of the 3? And would they perform better than an ethanol precipitation?

Maddie on Jul 14 2010, 09:41 PM said:

I read (somewhere on this forum) that if you add glycogen, it does help the precipitation but the pellet is at the bottom of the tube instead of on the side (because of the heavier weight). Is that true?
I can't use a speed vac unfortunately. I'd have to either pour or pipet the ethanol.

I still don't know which columns you are all refering to
I do a MinElute clean-up but it's not enough.

Also, how can I measure how much salt is in my extract?

Pito,
Oh, I know this kind of pain... It reminds me my lab couple of years ago... You can get free samples of various materials like dialysis tubing and SPE columns from companies but it may take some time to get it.

Maddie,
Ultrafiltration tubes - eg. Amicon Ultra-0.5 mL Centrifugal Filters for DNA Purification and Concentration

If there are people working with proteins in your institute/department/university they should have at least dialysis tubing, and may have ultrafiltration tubes. Oh, 4th method would be desalting/buffer exchange on Sephadex G-25 - also, protein people may have it.

I don't understand how a minelute column is insufficient. You can bind, then wash with PB as many times as you want, then wash with PE as many times as you want before elution. I suspect your problem is not with DNA purity, but rather with DNA quality. Is this genomci DNA? You may have problems with large DNA in minelute columns due to large size of the DNA.

You can perhaps improve the effective DNA quality by treatment with enzyme mixes to repair trashed DNA, such as pre-PCR (Epicentre or NEB).