Tips on PEG precipitation of DNA - (May/18/2008 )
Hey all,
This is a new technique for me and i didn't get great yields the first time around. I was interested in people's tips for this method. I use 1 volume of 1.6M NaCl/13% PEG-8000 solution, incubate on ice for 30 minutes, spin down for 15 minutes and ethanol precipitate. I've read to incubate in the -20C freezer after PEG addition so i'll do that. Anything else important?
Cheers, Rob
This is a new technique for me and i didn't get great yields the first time around. I was interested in people's tips for this method. I use 1 volume of 1.6M NaCl/13% PEG-8000 solution, incubate on ice for 30 minutes, spin down for 15 minutes and ethanol precipitate. I've read to incubate in the -20C freezer after PEG addition so i'll do that. Anything else important?
Cheers, Rob
If you are in a mood for browsing your answer, here are the links:
http://search.vadlo.com/b/q?keys=PEG+DNA&a...mp;sn=158621799
..
Thanks for the reply cellcounter. Unfortunately, like most of the information i've looked up on the web on this topic, there are only protocols in these links and no real explanation of why each step is being done or any tips.
Thanks, Rob
Thanks, Rob
Sorry about that. So far as I know PEG works as a crowding reagent, kind of facilitates DNA molecules coming together, which in turn speeds up and makes the precipitation process more efficient, by some laws of physics that were not required to study in my biology curriculum.
Salt and subzero temp facilitate ethanol mediated nucleic acid precipitation. If you were using isopropanol, you can do it at RT, in fact you should do it at RT, otherwise there would be lot of salt ppt. I am sorry for myself that I don't know the real mechanisms behind these lab-truths.
Thanks, Rob
Sorry about that. So far as I know PEG works as a crowding reagent, kind of facilitates DNA molecules coming together, which in turn speeds up and makes the precipitation process more efficient, by some laws of physics that were not required to study in my biology curriculum.
Salt and subzero temp facilitate ethanol mediated nucleic acid precipitation. If you were using isopropanol, you can do it at RT, in fact you should do it at RT, otherwise there would be lot of salt ppt. I am sorry for myself that I don't know the real mechanisms behind these lab-truths.
In our lab the most common reason for bad recovery is loosng of the pellet, PEG made the pellet relatively slimy and they don't adhere well to the bottom of the tube after the spin, so my advices will be take care of the way you get rid of the supernatant. I usually just invert the tube on a kim wipes soft tissue and then process immediately the sample.
If you still have a bit of ethanol left you can simply let it evaporate or incubate your tube briefly at 60°C to get the rest of the ethanol evaporated, be cautious not to verdry your pellet otherwise you will have hard time to resuspend it
Have you looked at the Molecular Cloning book from Sambrook? There must be a chapter about PEG precipitation (the 1. book, protocol 8 or 9). It might also give an idea.