> On 1 Aug 2000, Chunxin Wang wrote:
>> -Hi, all:
> -Does anybody know if the BAC DNA is methylated or not? One of my labmates
> -is trying to subclone one BAC into a binary vector. He cut the BAC with
> -Cla I, but it does not give rise to the right predicted digestion pattern.
> -He suspected that some sequences around Cla I sites has been methylated.
> -Is it true?
> Yes, I once had the same problem. The fact is that ClaI (which
recognises 5'-ATCGAT-3') is sensitive to dam methylation (which occurs at the A
residue in 5'-GATC-3' )
So, if the nucleotide immediatedly before a particular ClaI site is G, or
immediately after it is C, and you are growing the plasmid in a dam+ strain of
E. coli, it won't cut at that site. Solution - use a dam- strain!
Prof. Brian G. Forde
Department of Biological Sciences
Lancaster LA1 4YQ
tel. +44 (0)1524 594861 (direct line)
fax +44 (0)1524 843854
email b.g.forde at lancaster.ac.uk