Hi all,
We have been using the BioRad helium gun and been getting minimal
transformation efficiencies (0.6 um gold particles). In an effort to
optimize the transformation we are looking at several parmeters,
including the efficiency of DNA binding to the gold microcarriers. In
most experiments we get no more than 33% of the input DNA bound to the
microcarriers (for example 5 ug of DNA will result in 1.5 ug of DNA that
can be eluted from the microcarriers after the procedure). Is this a
normal number, or is there something that we should be checking (like
the spermidine).
In addition, if anyone would like to send the parameters that they use
(assuming that you're getting good transformations) we would like to
take a look at them and compare. Parameters that we are curious about
include; rupture disk strength, source of the rupture disk, level of the
algal plate for bombardment, how many cells on each plate for
bombardment, how long the cells are dried prior to bombardment, the
medium used for growth and plating, how long before results are seen,
any special treatment of the plates after bombardment (e.g. scraping off
the cells and replating at lower densities), tungsten vs gold particles,
the size of the particles, etc.
Thanks in advance.
Please send the information to dennisde at jmu.edu.
Regards,
Doug Dennis
James Madison University
