Screening for persence of restriction system

Duncan Clark Duncan at genesys.demon.co.uk
Fri Dec 16 11:51:23 EST 1994


In article: <warnick.183.000C10C0 at microbio.umass.edu>  warnick at microbio.umass.edu (Tom Warnick) writes:
> 
> I have been trying to transform a clostridium via electroporation. It works 
> fine as long as the DNA is from the same species, but doesn't work at all 
> using plasmids extracted from Bacillus; so I suspect that the clostridium has 
> a resrtiction nuclease system of some sort. Can anyone recomend a protocol or 
> a reference for screening for the presence of a restriction system and perhaps 
> also for further characterizing a restriction system?   Thanks,Tom Warnick

Many clostridia have been found to have type 2 RM systems. Screening is 
relatively straightforward.

Grow up your bugs from 10-100ml. Pellet cells and resuspend in 1/10 
volume 10mM KPi, pH 7.4, 0.1mM EDTA, 10% glycerol, 5mM mercaptoethanol 
(or 1mM DTT). Keep everything on ice. Break cells open by sonication or 
your preferred method (keeping cold!). Microfuge an aliquot and keep 
supernatant. Assay 10, 3 and 1ul of supernatant with 3 ug of DNA mix 
as below. Assay for 30 mins at 37C (unless you have a thermophile - 
if so use growth temp for assay). Add stopper/dye loading mix as per 
normal agarose gels and run on gel. What you are looking for is 
discrete bands showing a digested phage DNA mix. If you have a lot of 
non-specific nuclease then you will not see bands, just a smear. You 
will then need to adjust the amount you assay, low, medium, high or
acetate RE buffer and time etc. If it is still no good then the next
steps would be to run a sample on an FPLC monoQ or S column and
assay individual fractions etc first of all assay the crude extract
and let us know what happens.

For DNA mix use as many different phage DNA's as you can get hold of 
ie lambda DNA, unmethylated lambda, T7, T3, T5, AD2 etc. Use Medium 
Salt RE buffer. If you get clear bands then go back and reassay with each 
individual DNA. 

Good luck

Duncan   
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