I've been working with the GE NAP-5 columns for DNA desalting after digestion and precipitation. The first time I used the columns they worked just fine. Nevertheless, I've been trying to repeat the experiment and I've been unable to get my DNA from the column and the one I can recover is not really desalted. The first two attempts I used 100 uL sample (and 400 uL equilibration buffer, that is just DNA/RNAse free water), at first I thought this was the problem (because my successful recovery was done with 500 uL sample volume), so this last time I used a 500 uL sample. I recovered 14 100 uL elutions at aprox. 5 ng/uL each, and my sample concentration was 129 ng/uL.
I eluted using 100 uL steps, instead of 1 mL at a time. I think this could be my problem. Any thoughts?
Are you doing a phenol-extraction? Or are you directly precipitating the digest mixture? And are you monitoring nucleic acid content through A260nm readings?
Many small elutions would be problematic because the sample will diffuse within the column, which is why a constant flow is required i.e. by topping up as soon as the mobile phase enters the bed.
Thanks for all your answers.
Ted, I reproduced exactly my successful attempt, but suddenly it "stopped" working. My DNA is over 2k bp so I think it should be fine. In either case, I am repeating the procedure once again, exactly as I did the first time. Thanks!
Austin, I am directly precipitating my digestion mixture and I verify my DNA concentration through A260 nm readings using NanoDrop. I honestly think that the problem is the number and volume of the elutions, and that the first time it worked by a strike of luck or something. I'm gonna try with the whole mL at once next time. Thank you!
Andrei, yes, I do not reuse the column and I follow the given protocol. Hopefully it works next time. Thanks!
Hmmm...there are a few things then that may be contributing to your problems and making it harder to figure out what is going on:
1. Are you taking the supernatant after precipitation or the pellet? You should probably elaborate on this.
2. Generally, you cannot separate DNA and the restriction enzymes with these columns since the MWCO of these is on the order of 25 kDa. Rule of thumb is for good separation, you need to be 3-5x over the size cutoff.
3. even if there some separation, you cannot distinguish between the protein and DNA fractions, as proteins will also absorb at 260 nm.
4. The other issue is that if you are co-eluting, the enzyme outside of its optimal buffer may exhibit non-specific nuclease activity.
5. the time involved in eluting with 1 mL versus multiple 100 uL can be an issue as I mentioned previously. The best way to do smaller fractions is to count the number of drops that will give you a desired volume. Then you simply collect the desired number of drops in each tube.
Good luck, Sandra.
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