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8
(ii) Add 2 ml of the overnight culture to 50 ml LB medium in a 250-ml flask and shake
vigorously (250 rpm) at 28°C until the culture grows to an OD
600
of 0.5 to 1.0 (about 4-6 hrs).
(iii) Chill the culture on ice. Centrifuge the cell suspension at 3000xg for 5 min at 4°C.
(iv) Discard the supernatant solution. Resuspend the cells in 1 ml of 20 mM CaCl2 solution
(ice-cold). Dispense 0.1-ml aliquots into prechilled Eppendorf test tubes.
(v) Add about 3-5
g of plasmid DNA to the cells.
(vi) Freeze the cells in liquid nitrogen.
(vii) Thaw the cells by incubating the test tube in a 37°C water bath for 5 min.
(viii) Add 1-ml of LB medium to the tube and incubate at 28°C for 2-4 hrs with gentle shaking.
This period allows the bacteria to express the antibiotic resistance genes.
(ix) Centrifuge the tubes for 30 sec in an Eppendorf microfuge. Discard the supernatant
solution. Resuspend the cells in 0.1 ml LB medium per tube.
(x) Spread the cells on an LB agar plate containing 50 µg/ml kanamycin and 50 µg/ml
gentamycin (for pBIN-GW constructs) or 100 µg/ml spectinomycin and 50 µg/ml gentamycin (for
pMN-GW constructs). Incubate the plate at 28°C. Transformed colonies should appear in 2-3 days.
Note: After step (vi), the cells frozen in liquid nitrogen can be stored at -80°C. The frozen cells
can be used for future transformation experiments. Add about 3-5
g of DNA to the frozen cells and
follow the steps (vii) to (x).
2. Agrobacterium-mediated transformation of Arabidopsis
A. thaliana ecotype Columbia is genetically transformed with Agrobacterium using the
standard flower dip method (Clough et al. 1998) or its modified version (Kim et al. 2003).
(i) Plant 3-6 Arabidopsis seeds/pot in 6x6x6 cm pots. Let them grow for 5-6 weeks.
(ii) For pre-culture, inoculate Agrobacterium colonies/glycerol stock into 2 ml LB medium
with appropriate antibiotics. Grow overnight at 28
o
C.
(iii) Next day, around 5 pm, inoculate 0.4-1.0 ml of the overnight pre-culture into 200 ml YEP
containing appropriate antibiotics. Dipping can be done on the next day between 10 am and 4 pm,
depending on the growth of the Agrobacterium culture. For example, if you inoculated 1 ml of the pre-
culture, you can do the dipping in the morning.
(iv) To the 200 ml culture add 40 ml of water containing 12 g sucrose (final concentration 5%)
and 100 µl Silwett (final concentration 0.04%). Transfer the culture to a beaker and mix gently. The
“wonder mix” is ready for dipping. One 200 ml culture is enough for 2-3 pots containing 3-6 plants
each.
(v) Carefully take each pot containing the plants, dip them in the “wonder mix” solution for a
few seconds, transfer the pots to trays and keep them covered for overnight.
(vi) Remove the cover and let the plants grow and set seed.
Bibliography
Bahassi, E.M., O'Dea, M.H., Allali, N., Messens, J., Gellert, M. and Couturier, M. (1999) Interactions of CcdB
with DNA gyrase. Inactivation of GyrA, poisoning of the gyrase-DNA complex, and the antidote action
of CcdA. J. Biol. Chem. 274, 10936-10944.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation
of Arabidopsis thaliana. Plant J. 16, 735-743.
Doyle, T. and Botstein, D. (1996) Movement of yeast cortical actin cytoskeleton visualized in vivo. Proc. Natl.
Acad. Sci. USA 93, 3886-3891.
Griesbeck, O., G.S., B., Campbell, R.E., Zacharias, D.A. and Tsien, R.Y. (2001) Reducing the environmental
sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188-29194.
Kim, J.Y., Yuan, Z. and Jackson, D. (2003) Developmental regulation and significance of KNOX protein
trafficking in Arabidopsis. Development 130, 4351-4362.
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