Problems with biolistics protocol

Don Chen chend at ucs.orst.edu
Mon Mar 24 23:06:34 EST 1997


Hi,

We are attempting our hand in transformation of seed embryo callus
using established protocols.  We are having a problem with the
resuspension of the gold beads in water.  We find that the beads stick
to the sides of the wall of the microcentrifuge tubes and refuse to
make a pellet.  When washing with 100% EtOH, we experience no
problems.  We only have the problem when we add sterile water.  The
Bio-Rad people suggested that we should use Tekmar Treff (sp?) tubes,
we tried with no noticeable improvement.

People on campus use tungsten beads, and we are in the process of
trying them to see if we can get a pellet.  We can understand some
losses, however, it seems hard to imagine how anyone can quantify the
amount of DNA is being shot into the callus if there are significant
losses on the sides of the tube.

What are the experiences of those of you who have tried the biolistics
approach with gold beads?  Is it possible that the gold is from a bad
lot?  The tubes we are using are siliconized (US Scientific).  Could
these have an adverse effect?  One suggestion from the Bio-Rad tech
service was to use either a swinging bucket centrifuge or one of the
old style Eppendorf centifuges that mounted the tubes horizontally in
the rotor.  Could this help us get all of the gold into a pellet?

Any suggestions would be greatly appreciated.

Don 


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Don Chen            *  Standard disclaimers apply here.
USDA-ARS-NFSPRC     *
3450 SW Campus Way  *   
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