43 replies to this topic

[Maddie]

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

[K.B.]

Dialysis, ultrafiltration, solid phase extration.

[pito]

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?)

[K.B.]

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

[pito]

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.

[Maddie]

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?

[hobglobin]

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

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?

If you add some glycogen the pellet is better visible sometimes...and no speed-vac for drying...
anyway a normal column for a kit would do the job too, though there's always some loss...

[Maddie]

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?

[K.B.]

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.

[phage434]

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).

[pito]

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

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?

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.

[Maddie]

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.

[Maddie]

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  

[K.B.]

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

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?

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.

[Maddie]

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?