RNA contamination? - (Jun/19/2007 )

Hi everyone
I gave to my summer student a plasmid to transform in E.coli and to do a maxiprep from a single colony.
we use a commercial kit that always worked..but he manage to get the plasmid and an extraband at around 500bp as MW

All tha following digestion worked out fine but the bands still there..i was suggested it was a RNA contamination and to try to boil my sample to degrade RNA...smear of DNA but band still there.

any other suggestion??

thanks in advance

aleric4u

Hmmm is it RNA contamination or incomplete digestion?

Have you tried to just do a simple quantification using the ratio? 1.9 will gives you a rough idea that it is being contaminated with RNA.

If the RNase in the kit is old, it may not completely digest the RNA. Can you repeat the prep and add fresh RNase to test whether or not this is the problem?

Maybe your 500 bp band is just supercoiled plasmid dna (resistant to cleavage), but I'm not sure about that.

In this cases, show the picture would be very helpful to give a better opinion. I don’t think it is supercoild plasmid. 500 pb is too small. What is the size of you DNA and What is it for??? Is it a band or is it a smear? If it doesn’t hinder you, use it any way!