农杆菌培养与转化.doc

Transformation and storage of VIGS agrobacteria: Once your silencing vector sequence has been verified, transform a sample into electrocompetent agrobacteria, strain GV3101 (or try EHA105). Before transformation verify that the electroporator, electrocompetent agrobacteria cells, and appropriate selective plates are available. For a procedure on how to make and transform electrocompetent agrobacteria cells refer to p.122 of Weigel and Glazebrook’s “Arabidopsis: A Laboratory Manual”. Agrobacterium GV3101 has rifampicin resistance (at 25 ug/mL, chromosomal marker) and gentamycin resistance (at 50 ug/mL, Ti plasmid marker). The TRV2 and TRV1 vectors both confer kanamycin resistance at 50 ug/mL. (Agrobacterium EHA105 has background chromosomal resistance to rifampicin (25ug/mL) and chloramphenicol (25ug/mL), with no antibiotic marker on its Ti-plasmid.) For pouring new antibiotic plates and growing liquid cultures use the following antibiotic stocks and dilutions: Antibiotic Powder storage: Stock solution Final concentration uL stock/mL media Kan Room temp 50 mg/mL in water 50 ug/mL 1 Gn 4 deg C 50 mg/mL in water 50 ug/mL 1 Rif -20 deg C 25 mg/mL in methanol* 25 ug/mL 1 Store antibiotic stock solutions at -20 deg C. Make in aliquots and discard after repeated freeze-thaws. * do NOT use blue cap Falcon tubes for mixing methanol solutions (blue dye will dissolve into solution). PCR screen agro colonies (as previously done for e.coli) using 156F and 156R primers. Pick one colony with the correct insert size, and grow a 3 mL overnight culture. Use a sterile culture tube and shake overnight at about 270 rpm at room temperature in Gn50Kan50Rif25 LB media. Make a few glycerol stocks from this culture by doing the following: prepare 60% sterile glycerol in water by filter sterilization (do not autoclave glycerol solutions), and obtain a small quantity of liquid nitrogen. Label three sterile screw-cap plastic tubes. In each tube mix 0.333 mL of 60% glycerol solution and 1 m