Dear Clamy net,
To respond to an earlier request for parameters successfully
used in transforming Chlamy with the helium-driven biolistic device:
about a year ago I performed several transformations under the
following conditions: 1.0 um gold microcarriers, prepared as
recommended by the distributor (in Canada, Bio-Rad; I can send
a copy of their manual, which is *very* minimalist, to anyone
who wants one) using ethanol, 900 and 1100 psi rupture disks,
and placing the petri dish in the second position down from
the microcarrier launch assembly (the closest of the options
available on our machine). Under these conditions, and using
the nitrate reductase gene as a selectable marker and nit1- strains
with wild-type cell walls, I received 2-10 transformants per plate,
after plating 50-100 ul of liquid cultures at densities of roughly
0.5-1.0 million cells/ml. I found that I had to plate the liquid
aliquot about 2 hours before transformation and allow the culture
liquid to evaporate under a flow hood with the petri dish partly
uncovered, otherwise the liquid seemed to absorb too much of the
momentum of the gold particles and I got no transformants.
Lib Harris: when you post your results from transformation
experiments, I'd be interested to know what were your problems
with gold carriers. Sounds ominous.
Cliff Zeyl
B7JM at MUSICB.MCGILL.CA
