NTG mutagenesis protocols

Nick Waterfield nrw14 at mole.bio.cam.ac.uk
Fri Nov 20 05:32:31 EST 1998


Hi there.

Does anyone have a protocol (or reference) for Nitrosoguanidine (NTG)
mutagenesis of bacterial cells ? 

I am trying to chemically mutate Lactococcus lactis cells (gram+)  with
NTG but I have had a couple of problems;

i) I don't know the best ball-park concentration of NTG with which to
start doing "killing-curves". So far I have tried 100 ng/ml and 500 ng/ml
for up to 2 hours. Neither killed any of the cells. How much higher should
I go ?

ii) I don't know the best way to solubilise NTG. I have been making weak
solutions of NTG (800 micrograms / ml) in SC [0.15M NaCl,  0.01M Na
Citrate pH7] by dissolving at 55'C. As NTG is very sensitive I was
concerned this may inactivate it. Also DMSO solubilises NTG but would this
inactivate it?

Thanks in advance.


Nick Waterfield
Cambridge Uni
Pathology Dept.
nrw14 at mole.bio.cam.ac.uk



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