RNase treatment of plasmid - (Dec/07/2005 )

after miniprep (for plamid DNA isolation) im facing a problem of presence of RNA. when i perform RNase treatment to remove that and afterwards digest the product with restriction endonuclease (in order to confirm the insert size befor sequencing) i have to see a smear only...like that of degraded DNA.... ...
nothing is wrong with RNase...
i am after a small gene (300bp)..so RNase treatment is needed before restriction digestion...i think so......
plz help!

any one who can help??
thnks!

From your post I am not sure at what point you are adding your RNAse to your prep.

From my experience, it is best to add it to your bacterial reusupension buffer (prior to lysis of bacteria).

When I do this and then do alkali lysis and ethanol precipitation, I have not seen any degraded RNA in my preps and they are just fine for restriction digest.

thanks bluescript
i am adding RNase after absolute alcohol precipitating and dissolving plasmid DNA in TE or water, before restriction digestion
i will try wt u have done

After alcohol precipitation and 70% wash, dry the pellet and resuspend in autoclaved water containg 40ug/ml RNase.(though 20ug/ml is enough). Either keep for 30' at RT or 37C. This works fine for me.

thank u Ruch & Molonco...
i dissolve plasmid DNA in TE buffer & then add RNase to it.....
is there any difference b/w dissolving the DNA in TE or autoclved water before subjecting to this treatment?
i still have not followed the suggestion given by bluescript...i will do miniprep tomorrow....tell me what to do???

adding RNase prior to alkai lysis solution do not work in this case...how could it be?? as RNase will have to remove RNA after lysis...
any other suggestion bluescript???