RNAse and DNA digestion in Southern Blot - (Nov/30/2006 )
I'm going to do southern blot by using genomic DNA extracted from cells.
1. Should I remove the RNA from DNA before I do southern blot?
2. If I should, when is the best time to do the removing, during extracting of the DNA or loading the gel?
3. And the only RNAse I got is from Sigma (cat#R5500). According to the manual of this product, it is not DNAse free. They do give out a method to get rid of the DNAse. But it is too complicated. Is there any simply and reliable method to remove the DNAse from the RNAse.
4. I know that the DNA should be digested, before the southern blot. Is there any special concerning about the digestion, such as the size of the digested fragments?
If anyone can help me out of this, I will appreciate that help very much.
i can remember my first 6 months of my phd course from ur topic.
well u should perform RNase treatment when u prepare the genomic DNA.
if u remove RNA in the time of prepare DNA sample then there is no need for further treatment for RNA. usually i add some amount of RNase in the extracted DNA and incubate it for 30 to 60 mins.
DNA digestion is must for southern blotting and choice of enzyme is depend on ur particular purpose.
U shud remove RNA before southern. u can add RNase before ur Phenol:chloroform treatment during extraction, it helps in avoiding further cleaning of the DNA.
if u do not feel fine to use ur RNAse just as it is then u may heat it in water bath @ 80 C. for 1 hr (it denature the contaminating DNAse from it).
regarding the DNA digestion- digest till u see an uniform smear from top to bottom of the gel. this is good enough for southerns.
hope this is fine..........!
Thanks for your nice suggestion.