Methods for desalting - (Jul/14/2010 )

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

its normal to lose some DNA after enthanolprecipitation. I have that always.

Do keep in mind that when you see a pellet that this means you have a lot of "dirt" . Pure DNA is not visible. So I do not know if you really lost DNA or just dirt?
(did you measure DNA quantities before and after ethanolprecipitation?)

I always use ethanolprecipitation to clean my samples and it works just fine.
What I do is right before the ethanolprecipitation is the add 2 samples together in 1 tube.
I always measure the amount of DNA with a nanodrop before I do the ethanolprecipitation and then I see wich samples I can add together.

@ K.B.

you are offcourse right, but as Maddie allready said: she doenst have those things and needs to look for them.
And yeah, you are right, there are a lot of free samples there, but the question remains: how many time does she need to do it? Samples will run out and in the end she might need to buy something.. and without money..

@ phage434

I have had a similar problem. I couldnt get a nice insert and in the end it all came down to the fact that the DNA wasnt pure enough... a simple ethanolprecipitation fixed the problem.

OK, I will expand. I extract powdered bone in 4ml EDTA 0.5M (+0.5% detergent+ proK). After an overnight incubation that decalcifies entirely the sample, I concentrate in an ultra-4 down to 100ul. Then I transfer in an eppendorf and clean up with the MinElute (once or twice depending on the application).
The reason I think I still have salt is because my Absorbances suck. The A320 are around 0.15 and the A260/A280 are far below 1.8. I will actually try the PCR repair kit as I got 5 free samples (thanks again to the person who recommended it on another discussion). Then I will do a next generation library.
The first time I did a library with a "regular ancient DNA extract", the library didn't turn out so good.

When I amplify STR with my extracts, I can use up to 5-10ul in a 25ul reaction if I clean up my extract twice. 30ul of extract would inhibit any PCR reaction (30ul is what I'm supposed to use for the library prep). So I'm thinking that if 30ul of extract inhibit a polymerase, they might as well inhibit the Klenow, ligase, kinase I used for the library prep. Ideally, I would feel more comfortable with a good Abs ratio (even if it's not exactly 1.8). Currently it's closer to 1.3.

Pito: I haven't tested the precipitation on my extract yet. It's on my plate for today
I'll let you know how that turns out.
At least I have lot of sample material, so I could extract, precipitate and then pool many "clean" extracts (I guess) for making a library.


KB: thanks a lot for the link. Can I do something similar with my ultra4? What if I add water or TrisHCl instead of NaCl 10mM? I could use a ultra4 10kDa not to lose too much DNA. Bascically, you simply dilute the salts that way, right?

I think I will try both methods today (dilution and precipitation) and we'll see what works best.

Hmm I forgot an important detail .
The extracts are full of humic acids that contain some pretty big molecules that get retained when I concentrate with an ultra4 30 or 50KDa. Can't use a 100kDa filter because my small DNA fragments would go through. So it's very likely that humic acids contribute to messing up my absorbances.
Then diluting wouldn't work.
But ethanol precipitation would...hmmm, pito? looks like I can run but I can't hide

Maddie on Jul 15 2010, 05:31 PM said:

Aha! So you do know and use ultrafiltration!
Yes, you can use water or tris. No, it's not simple dilution - salts are not retained by membrane and are removed and if you do this right, your compound of interest won't be too much diluted, you can even get it concentrated.

Looks like I am! Some new hidden talent

OK, now I need to find a way to stick a centrifuge in the fridge since I don't have a refrigerated one.
How bad is it to centrifuge the extract+ ethanol at RT?

Why dont you test the 260/230 ratio if the samples are rich of humus?

If the ethanol is cold when you add to your samples its ok.
I never centrifuge my samples in a fridge or a centrifuge you can refrigerate.

I am. The A260/A230 is around 0.10 to 0.15. Shouldn't it be >1.5?

I found a refrigerated centrifuge in my biophysics neighbor's lab .
Still waiting for my sodium acetate and I'll roll.

If you still have a humic acid problem this paper might help:
D. Sutlovic, M. Definis Gojanovic, & S. Andelinovic: Rapid Extraction of Human DNA Containing Humic Acid.
Croatica Chemica Acta 80 (1) 117-120 (2007).
They added polyvinil-polypyrrolidone to avoid TaqPolymerase inhibition.
(The names are a bit different with Slavic letters, I cannot write here)

Maddie on Jul 16 2010, 07:49 PM said:

didnt you mention it was the a320 that was about 0.15?


Well good luck with it and the paper Hobglobin mentions, might be helpfull.

Oh this is funny. I read that paper yesterday. It's interesting, they show that humic acids have an impact on several kind of enzymes. However, they don't suggest anything to get rid of them .
We all want to get rid of them but they bind to silica, they seem to precipitate during an ethanol prec...they are really a bother. When working with ancient DNA, you don't want to make too many handy work because of the contamination issues. So most protocols that could help remove the HA aren't really recommended for people working with small amounts of human DNA. I found a very interesting paper on HA yesterday where they show how to comfirm the presence of HA with fluorescence. Since my buddies in biophysics have the instrument, maybe I'll try to measure what's left in my extracts.