Miniprep SNAFU

[john brennand]

mmmm...

My 2p worth

A properly done miniprep shouldn't contain any genomic DNA.  So (you 
dont specify) is the smear just normally digested genomic DNA that is 
obscuring any plasmid bands ?

In any case I would make solutions I,II,III up fresh and try again.

What happens if you incubate the miniprep (no additions) at 37o for an 
hour and then run the gel ?  If it degrades then you have DNAse 
contamination that is only being activated upon incubation at higher 
temp - if that is the case, phenol or Strataclean the minipreps.

Does DNAse need salt to work ? ie, if you add salt (buffer) to your 
miniprep and incubate at 37o does that accelerate degradation ?

If you are losing the plasmid DNA only upon incubation with an enzyme, I 
would suspect contamination of the enzymes - or more likely, the 
incubation buffer, water, tubes, tips, etc.


Hope this helps.

john

PS like most people, we now use kits by Promega, Qiagen or Bio-Rad and 
have never had any problems - a good investment compared to the cost of 
people's time in trying to sort things like this out.