A way to avoid insoluble precipitate with sodium acetate - (Sep/04/2008 )

Hi there,

I routinely do DNA extraction from gels using a gel nebulizer kit, after whitch I have to precipitate my DNA with sodium acetate, in order to concentrate it in a smaller volume. Now after precipitation, I always get an insoluble pellet (from whitch I can still recover DNA).

1. Am I alone is this frightening world of insoluble precipitate?
2. Are the residual salts inhibiting subsequent enzymatic reactions?

-Madrius-

I have had insoluble material before when extracting gDNA from tumour material before, this was because I overdried it, could you be doing that (I doubt that but it is the most obvious thing I could think of) ? I still got usable DNA and when I digested it there were no obvious problems.

-stevo-

The pellet is most certainly not overdried. And I can recover sufficient DNA using this method, and it can be redigested afterwards. The enzymatic reaction I'm most worried about is ligation (even if I had some positive clones in the past).

Thanks for your input though

-Madrius-

Do you wash the pellet with 70%EtOh?? This help to get rid of the NaAc and "rehidrate a little bit the DNA.

-merlav-

Yes, I do wash my pellet with 70% etoh.

Strange thing..

-Madrius-

QUOTE (Madrius @ Sep 5 2008, 10:25 AM)

Yes, I do wash my pellet with 70% etoh.

Strange thing..

Madrius, Did you see this insoluble stuff using any other kit?

-scolix-

Hmm, i could not tell you being 100% sure, but I guess so.

It seems that the salt stay as a precipitated form..

-Madrius-

If you can borrow 1-2 columns from another kit, try gel extraction and check it out. Just a suggestion !!!

-scolix-

Nanodrop that stuff see how well it turns out perhaps.

Normally gel extraction give me a problem of high salt contamination.
bad for ligation for sure.

-Hanming86-

Thanks for the inputs. I'll check that.

-Madrius-