农杆菌转化(国外英文资料).docVIP

农杆菌转化(国外英文资料) Plant expression vector; transformation; Agrobacterium tumefaciens Part 1: Agrobacterium mediated transformation of rice 1, Agrobacterium selection: LBA4404, EHA105, GV3101 2. Agrobacterium activation: draw the preserved Agrobacterium on a solid LB medium (or with or without antibiotics, LBA4404:Rif or Str; EHA105:Rif or Str; GV3103: gentamicin). If no antibiotics were added, the Ti plasmid could be lost, resulting in the lack of infectivity of the Agrobacterium tumefaciens, and the concentration of antibiotics was 50 g/ml. Culture at 28 DEG C. 3. Preparation of competent cell of Agrobacterium tumefaciens: 1) were single colony was inoculated in 3ml LB medium in 220rpm 28 DEG C to OD600 =0.5 medium. 2) absorb 1.5ml bacteria in the centrifuge tube, and 10min in the ice bath; 3) 5000 (13000) RPM centrifugation 30s, discarding the supernatant; 4) precipitation with 1.5 ml 0.5M NaCl suspension, ice bath 20min; 5) 5000 (13000) RPM centrifugation 30s, discarding the supernatant; 6) each with 100 L 20mMCaCl2 suspension for conversion; The prepared sensory cells can be used immediately, and can be packed in sterile centrifuge tubes at 200ul per tube for 4 hours and stored for 48 hours. After long storage, they have to be frozen in liquid nitrogen and stored at 70 DEG C for a long time. When used, remove from a temperature of 70 degrees and place it on the ice. 4 and DNA direct transformation of Agrobacterium tumefaciens: 1) plasmid DNA 0.1 ~ 1 mu g (5 10ul) was added into the competent cell of 50 L Agrobacterium, and then 30 min in ice bath; 2) put it in liquid nitrogen 5min (or 1min), and then put it into the water bath of 37 DEG 5min at once; 3) take out the centrifuge tube, add 0.5mlLB, and train for 3 to 5hr at 28 DEG C and 220rpm; 4) take out the bacterial fluid and smear the plates on the LB plates containing the antibiotics, then invert them in the incubator at 28 degrees centigrade. About 2 days, the colonies are visible. (pEmu: AMP+Rif/Str; pK: carrie