6 replies to this topic

[felixwang]

I did a large prep using PEG Precipitation method. I added ethanol and NaOAc to precipitate DNA. After centrifuge, I got a DNA pellet and washed it with 70% ethanol twice. Then I left the tube open to dry at room temperature for about two hours. It seems that the DNA became over-dried and very very hard to dissolve. It formed a transparent pellet in solution. I put the DNA solution at 37 for three hours and there was no effect at all. How can I dissolve the over-dried DNA?

[methylnick]

felix,

what have you dissolved the pellet in?

Nick

[yongyk]

Dear Felix,

I guess you dissolved your plasmid pellet in ddH2O, right?
Try to dissolve in 8mM NaOH.

Next time when you extract any of your DNA, never never let it over dry.
After you discard the supernatant (70% ethanol) try to pipet out the what ever solution remain at the bottum of the tube and let it air dry brifly. Or you can put on thermal cycler block, set at 50-60oC for 2 min (I always do so).

Regards
Yong

[felixwang]

Thank you very much for your help! I dissolved the over-dried pellet in ddH2O. Because I am going to use the DNA in transfecting cells, I don't want to add NaOH to the solution. Can I simply spin down the un-soluble pellet and use the solved DNA solution (which is enough for transfection)?

[fred_33]

hi
hum when nucleic acid forms an unsoluble pellet (lokks like a gel?) it's probably RNA. If you want to check it, try to dissolve it in a small amount of water (150µl ddH2O) and heat it to 60° duting 10'. If dissolving well, check on agarose gel.

[chandima]

hi,
when there is lot of DNA it may take time to dissolve. I usually leave it at 4 C overnight. Better not to disturb it by vortexing and trying to brake the clumps etc. if it is DNA should  has to dissolve, isnn't it?
chandima

[george@CASE]

to add to this, DNA pellets dissolve better in alkaline solution. So check the pH of your water, or use 10mM Tris-Cl, pH 8.5 aka buffer EB.