Trond Erik Vee Aune wrote:
> We have general problems with cloning and manipulations of Bacillus DNA
> (AT-rich!) in E. coli (DH5 alpha), related to DNA instability, difficult
> to clone, etc. We would be happy to know if anyone knows certain general
> tricks (appropriate E. coli host, growth temperature, DNA preparation,
> etc) that is useful when working with such DNA??
>> trygve
Yeah, I work with Brachyspira which also has a low G+C content (around
24% in coding regions, as low as 7% in non-coding regions).
For expression work, try using the CodonPlus-RIL strain. Just do a
search on the web for "CodonPlus RIL" and you'll find it. Also try
looking up a couple of E. coli strains called "C41" and "C43". You can
find them in the literature if you do a search. I don't have the
reference on hand at the moment.
Most molecular biology techniques are the same, however. DNA preps are
the same. PCR is usually troublesome, often requiring annealing
temperatures down as low as 48oC, and careful optimisation of primers.
Cloning isnt usually too hard, but of course you need to pick your
enzymes carefully because (aside from methylation) you are not going to
be able to use a lot of them because they're recognition sites are too
G+C rich.
If you're doing protein work, then the growth conditions you use will
depend on the protein you're trying to express. It's not really G+C
dependant.
Scott.
