vacuum needed? more replies

Niki Robertson niki_robertson at ncsu.edu
Tue Dec 3 13:17:20 EST 1996


Dear netters,
Here are two more responses to my query as to whether a vacuum was
actually necessary for transformation.  It sounds like it would be quite
worthwhile to test transformation without the vacuum, making sure to
include Silwet or detergent.



Our uncontrolled data on transforming Landsberg erecta with and without a
vacuum (but including the "vac-in" detergent from Lehle) were:

with vac:     1.0% +/- 1.1%   kanamycin resistant seed per plant
without vac:  1.4% +/- 2.5%   kanamycin resistant seed per plant

Thus it works fine, but is somewhat variable, so it is wise to do a few
plants per construct.   We are using kanamycin as a marker, and this is
data taken from 4 different constructs in 3 different vectors.  We have
confirmed many of the transformants via GUS staining and/or Southerns.

We follow the modified protocol from Andrew Bent, more or less.
Hope this is helpful
Rob Martienssen


I have tried omitting the vacuum, actually with rather good results.  
I haven't done enough to make comparison statistically valid, but it 
actually worked better in my hands than with the vacuum.  I asked 
Nicole Bechtold about their trying it, but her group don't use silwet 
at all, which probably makes a big difference if you are omitting 
the vacuum.  so it is well worth a try, but include silwet in the 
infiltration medium.
Justin Goodrich
ICMB


Many thanks to Rob and Justin for sharing their results.

Niki Robertson
niki_robertson at ncsu.edu



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