Difficulty resuspending DNA after precipitation - (Feb/28/2008 )
Has anyone got any tips here? I think i heated my sample too much at the end and apparently that makes it more difficult to resuspend the DNA. I also resuspended in water, will Tris buffer resuspend the DNA? I tried heating my sample with the water in there but that didn't help much.
I assume your sample is genomic or plasmid DNA after some kind of prep. Most DNA I have worked with will resuspend if you leave it in water at 65oC for an hour or so or if you put it in the fridge overnight. Also, have you run your DNA on a gel to see if any had redissolved, sometimes you just get precipitate during purifications that dissolve badly but are not DNA?
I hope you have learned a lesson from this experience.
But first of all DNA for storage has to be kept in TE. Leaving it in water is not a good idea.
DNA is more soluble in slightly alkaline solution, but I don't know if that will help should the DNA pellet be that badly over dried.
Try leaving your pellet in TE on a heating block at 68 Celsius for 10minutes. Then take a pippett and break up the pellet into small pieces. Leave to reheat again. Repeat pipetting the solution up and down. Repeat heating and pipetting until the pellet goes back into solution. (This process will badly damage plasmids in the 30kb range and above.)
Best case, all the DNA will go back into solution. Worst case, the pellet remains but you will get the DNA back into solution, although at a reduced concentration.
Thanks for the replies guys, no thanks for the condescending first sentence Pernese - just asking for advice, not a telling off thanks. It is plasmid DNA, just 8 kb or so. I got a low OD260 reading for the solution (not containing precipitate) so i suspect most of the DNA is still precipitated. I'll try heating the DNA at 65C for a while and see how that goes, i only heated it at 37C last time so that may not be enough. If that doesn't work i'll use some Tris buffer. With regards to Tris, i usually resuspend in Tris.Cl pH 8.5, but not in buffer TE because EDTA may affect my subsequent reactions. Furthermore, i wanted to ensure that the Tris in my buffer did not have any toxic effects on my mammalian cells during transfection, however small that effect may be. Thirdly, i needed an endotoxin-free solution and only had endotoxin-free water available. So there was a few reasons i went with the water over Tris for resuspension.
My appologies killerkoz17. It was not my intend to sound condescending.
If the DNA is to be stories and Tris is inappropriate, consider HEPES buffer as an alternative.
Keep at the heating/pipetting. It is frustrating work, but the DNA pellet will go back into solution.
No worries. Cheers for the suggestions. The heating and Tris should solve the problem but if not i'll go with HEPES.
If I am going to use the plasmid for transfection, I usually resuspend it in 0.1xTE, just to keep the pH. The cells would be still okay after transfection.
Take care while precipitating, Sometimes if DNA is over dried, then its difficult to resuspend it.