Problems with biolistics protocol

Kimberley Snowden kcsnowden at ucdavis.edu
Thu Mar 27 14:59:24 EST 1997


Do you need to resuspend your gold in water?  When I did this I
resuspended in ethanol and then spread the gold on the macrocarrier,
and allowed the ethanol to evaporate off, before assembling everything
and shooting the tissue.  
Kim

chend at ucs.orst.edu (Don Chen) wrote:

>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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