I have a DNA/TE buffer solution, the molecular weight of the DNA is 185 kb and this solution has some lipids and protein in it (from lysis of E-coli). The dialysis tubing I used is 300,000 MWCO. I put 40 ml of DNA solution into this tubing and put it in 5 L TE buffer for 3 hours, after which I changed the buffer and let it be there overnight. Then I did a NanoDrop to see the concentration, unfortunately the DNA almost disappeared. Why did the DNA almost disappear? Do you know what the problem could be?
if I understand correctly the dialyzer you used had a molecular weight cut-off of 300,000 daltons (seems really high to me), so a molecule with molecular weight of 185,000 daltons would be able to freely pass thru it
or maybe I am misunderstanding your question
Tens of thousands of daltons would still easily pass thru a pore which allows 300,000 daltons to pass
Why are you dialysing the DNA? Using a DNA extraction kit you can have the whole procedure done in a few hrs with the sample eluted in TE. The added advantage is that the eluted volume will be much less that 40ml.
You seem confused its either 185 kbp or 185,000 kbp can't be both. If it's 185 kbp and the MW of a base is about 600 daltons then the MW of you DNA is 600x185,000, which is 111000kDa so no issue with tubing. But have you considered the DNA sticking to the tubing
The other consideration is that the dialysis membrane is contaminated with DNase. Depending on the material, heat treating it (boiling) might help, otherwise, making sure there is some type of DNase inhibitor on both sides of the membrane might be helpful.
Have you considered loss due to DNases/Nucleases? I don't know what your end-point analysis is but I definately would try a different technique to Isolate the DNA from your bacteria. Try using a Kit maybe. The chances of Nucleases degrading your DNA is the dialysis tubing is just really high. You can try to use DNase Inhibitors in your solution too. Otherwise, I agree with Matthew Lalonde on trying the Phenol-chloroform extraction too.
Is it possible for the DNA to leak through the tubing closure during dialysis? Also, if the tubing dries a little bit during the filling process, will that make the pores larger or even broken? Also, the tubing I received was soaked in sodium azide, which is a bacteria-cide, before dialysis, I did not use D.I. water to rinse it clear, will this matter?
Dialysis of DNA is not good practice since you cannot consider the DNA a spherical molecule like proteins. Then the cutoff of the membrane is not a good indicator.
To clean the DNA the usual procedure is to go through a gel filtration column, G25 or G50, eluted with water or the new buffer. The gel filtration can then be used either to remove or to exchange the buffer. If you don't want to prepare the column you can buy prepacked column like NAP from GE.
The DNA can then be concentrated by liofilization.
Dialysis is used by NEB to get highly pure lambda DNA, so it is obviously OK to do this if you have the time. Of course there are other much faster ways to clean up DNA for most purposes. You should assay your DNA before and after dialysis using picogreen to see if you are really losing it. Dialysis is probably removing a lot of stuff that absorbs at 260 nm if that is how you are quantifying it. Unless you have sheared the crap out of it, your DNA will not pass thru the membrane. I would suspect there wasn't much DNA to begin with. DNases will not function in TE buffer, so no problem there.
Hello, Yanfei Li, did you find the cause of your problem? I do have similar problem and I try to find the possible causes of my problem in your story...
I would like to share my thoughts on this question. I have tested 12 KDa MWCO membranes to remove salts from DNA extracts obtained from marine sediment samples. I can guarantee DNA won't cross the membrane after 2 hours at room temperature, neither overnight at 4 C.
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