I extractd BAC plasmid for FISH probe labeling. I refered to classical alkaline-SDS lysis method and found that the RNA contamination in BAC is much abandunt than usual small plasmid extract. In order to remove the RNA, I used RNase A to digest it at 60 oC， or LiCl to precipitate it. However, the RNA could't be removed completely. Did anyone experience this situation? Will the RNA contamination disturb the probe labeling efficiency using nick translation methods?
Thanks!
I would be carefully. RNA should not survive an RNase A treatment. You may try a column purification, e. g. the NucleoBond Kit.
see RG discussion: https://www.researchgate.net/post/Which_is_the_preferred_method_of_BAC_DNA_isolation
Hi Hu,
I do not think that RNA contamination could influence BAC-probe labelling using nick translation since it is normally an excess of all components (enzymes, dNTP mix) in the labelling reaction.
Best regards,
Frantisek
I've done BAC DNA labeling with RNA contamination plenty of times. At least with 500 BAC-clones. It always worked well and I've never bother myself with RNAse treatment (I'm talking about large numbers of samples in 96-well plates, which we got from robot extractions). Only problem with RNA contamination - it affects Nano-drop values, so you never know how much DNA you're taking in labeling. From my experience with robotic samples Nano-drop conc. can show you crazy values - some 6 ug/ug - but in fact you have 1 ug in about 10 ul - so you need to figure out what will work for you empirically. Regarding to self-extraction, purchased kits usually give pretty good yield without contamination, even if you have little RNA - it's not a problem, at all.
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