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Tetracycline for transformed Agrobacterium

Nobody nobody at hgmp.mrc.ac.uk
Wed Mar 28 19:18:51 EST 2001

Dear Arabidopsis Researchers,

I would like to thank all those who made comments and suggestions to=20
my inquiry.
Here is a summary of the suggestions I received.

1. To switch to an other binary vector with kanamycin resistance.

2. Steve Farrand's lab has developed an Agrobacterium strain that=20
will not form spontaneous tetracycline resistant colonies. The=20
reference is:
Luo ZQ, Clemente TE, Farrand SK. 2001. Construction of a derivative=20
of Agrobacterium tumefaciens C58 that does not mutate to tetracycline=20
resistance. Mol Plant Microbe Interact. 14(1):98-103.

3. By mating my cosmids into GV3101 using a helper E. coli strain=20
(HB101 pRK2013).
A simple protocol is to spread cells onto an LB plate together, at a=20
ratio of 2:1:1 GV3101:helper:donor E. coli, grow overnight at 28C,=20
then collect cells in around 3 mls liquid LB and make serial=20
dilutions to plate on selective media. If you want, you can select=20
for cells that don't contain the helper plasmid, but I didn't bother.=20
DO select with something that will remove the mixed in E. coli, like=20

As my Arabidopsis mutant with constitutive expression of GST1-GUS has=20
kanamycin-resistant gene, I cannot use the selection marker. I chose=20
the third method because there are helper strain and donor strain in=20
our lab and the professor Steve Farrand also recommended that method.

Thanks again and good luck.

Jong Tae Song
jtsongg at hanmail.net
Molecular Genetics and Cell Biology, The University of Chicago

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