>> Can someone provide me with a tried and trusted method for
> getting phage DNA from lambda EMBL3? I've tried plate lysates in
> the past, but often got large amounts of contaminating
> nucleic acids, making it difficult to do mapping or subcloning.
> Andy Phillips (PHILLIPSA at AFRC.AC.UK)
Contaminating nucleic acids are usually due to unsufficient treatment with
DNAse/RNAse before digesting the phage coat with proteinase K or
phenol/CHCl3. You probably need to add a lot more DNAse at the initial step
to completely digest bacterial DNA which later on you can impossibly get rid
of. I usually use about 50ug/ml of DNAse/RNAse at least (as opposed to 1ug/ml
recommended in a protocol in Sambrooks ) and incubate 37C for 1hr. It always
works fine. Even too much DNAse can't hurt at that stage because it can't do
any harm to phage DNA which is still in the coat.
antje at menzies.su.edu.au
Menzies School of Health Research