Methods for desalting - (Jul/14/2010 )
Here is what I got for 3 extracts (ethanol precipitated if I may say that) that all gave DNA concentrations under 5ng/ul
what was the DNA concentration prior to ethanolprecipitation?
Those results do suck lol
No, this is after the ethanol precipitation. BUT it was even worse before
Yeah, I know, but did you measure it before?
Did you measure the DNA quantities before eth.precipitation? You need to do that.
Oh yes I had
And for DNA concentration:
sample 1: 5.3 to 1.5 (ouch)
sample 2: 3.9 to 4.3
sample 3: 4.7 to 2.3
Here is what I read about the nanodrop:
The manufacture-stated dynamic range for the NanoDrop
instrument is large: 2 - 3,700 ng/μl of dsDNA. In practice,
GenVault finds that DNA concentrations of less than 10
ng/μl are seldom reliable as the basis for A260-based
DNA concentration determination (see Fluorometric
measurements section and Table 3 for explanation), and
DNA concentrations of less than 20 ng/μl are seldom reliable
as the basis for the more demanding analysis of DNA purity
via A260/A280 or A260/A230 ratios.
This could explain my bad ratios maybe.
Yeah, you are working with small amounts, so its hard.
But to be honest, what I would have done is added the samples together.
And the first one, that you lose so much DNA is strange.
PS. you didnt really improve a lot ... its sounds strange to be honest.
What protocol did you use for ethanolprecipitation?
Here is the protocol I used (found on Bioforum )
I don't have glycerol unfortunately.
50ul of extract
+ 130ul cold Ethanol 100%
+ 5ul Sodium acetate 3M pH 5.2
Mix (I didn't dare to vortex. Ancient DNA is damaged enough already)
Place in -80°C Freezer for 2 hours
Centrifuge for 30 min. at 4°C (more than 12,000g)
Remove alcohol without disturbing the pellet.
Add 200ul of 70% ethanol (-20°C) and centrifuge for 5 -10 min at 4°C.
Pipette out all the remaining alcohol
Centrifuge briefly for a few seconds and pipette out all the remaining liquid.
(Let it dry with open lid)
Add warm TE/water and wait for 2-5 min. and then mix and centrifuge.
For sample 1, I probably pipetted most of the pellet.
I will try again with other samples.
I could pool several extracts of a sample together and that would increase the A260. I'm afraid it would also increase the A230 a lot though.
Next week I will try this protocol I mentioned on the other posts and will clean up DNA with silica beads. I hope it will help.
How do you remove the ethanol?
Do be honest: I would certainly not pipette out the alcohol in the second stage... NEVER
And why do you need the glycerol?
I never use that.
Yes, I confess father, I did use the pipette (bad Maddie). Although in my previous life, I was just flipping the tube. I guess that's how you do it, right?
OK, OK, I will start all over again with new extracts.
The glycerol is supposed to help when you precipitate very small amounts of nucleic acids (I read that here as well).
Sooo what's the magic trick to quick a.. with an ethanol precipitation?
Eum I can see why you add the glycerol (I have seen that here too) but I never did it.
Anyway: like you said: just put the tube upside down on a sheet of paper to let it dry. Let it stand upside down for maybe 40 minutes, then flip it and let it stay for another 40 minutes or so.
(before flipping it back , make sure to remove the moisture at the side of the tube (the drupplet of fluid thats hanging there.. if not, even better)
And the magic trick is to be very cautious. And before you measure the DNA on the nanodrop: do not spin it down. You might spin down the DNA so much that when you measure you dont measure it since you only pipetted water or whatever...
and when you see you are low on DNA after the first step (before the eth. precipitation) you might wanna add 2 samples together.