Purification of Double stranded radiolabelled DNA - Purification problems (Feb/23/2005 )
Hello.. can anyone help:
I am starting to read up about how to do an EMSA assay and was given a protocol to look at. I am just a little puzzled as to why for the purification step after labelling annealed DNA probe with 5' 32P, we add t-RNA? I can't find any information about this on the web. I would appreciate it if anyone knows what this is for and what type of t-RNA am I meant to use/where to get hold of some? Thank you!
Hi. I suppose that you should add tRNA to help precipitate the labeled DNA, as its concentration will be too low. tRNA acts as a carrier in these circumstances. I've never used tRNa for precipitation, though, I use glycogen, which is another possibility.
When I label the oligos for EMSA, what I do is clean them with a sephadex colum instead of precipitating them.
Hope that it helps!
Here, I've just checked for some information on the web:
Coprecipitants are inert substances used to aid recovery of nucleic acids during alcohol precipitations. While they can be used for precipitating large amounts of nucleic acids, they are essential for quantitative recovery of small amounts of nucleic acids in dilute solutions. Often, the use of such molecules is desirable for no other reason but visualization of the pelleted precipitate after centrifugation.
Purified Torulla yeast RNA is suitable as a coprecipitant in nucleic acid precipitations. It cannot be used in reactions inhibited by exogenous RNA, and it is the most inexpensive source of a high quality coprecipitant. This total RNA preparation consists of 300-500 base fragments and is supplied at a concentration of 5 mg/ml in diethyl pyrocarbonate-treated distilled water. It is typically used at a working concentration of 10-20 µg/ml.
Another useful coprecipitant is yeast tRNA purified from brewer's yeast. When used at a final concentration of 10-20 µg/ml yeast tRNA is an effective coprecipitant to aid in recovery of small amounts of nucleic acids
Thats brilliant! thank you for the info its great help