Quint Lab:arabidopsis protoplast transformation

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arabidopsis protoplast transformation Advice Pipette protoplasts only with a cut pipette tip Keep all solutions and protoplasts sterile all solutions should have room temperature (long time storage at 4°C) Preparation 5 days before: order tissue culture from Silvia (ca. 40 ml is enough for at least 20 transfection reactions; e.g. Col-0 d) 1 day before: prepare all solutions Preparation of cell culture zentrifuge 5 min at 800 rpm carefully decant supernatant resuspend pellet in 30 ml 0.24 M CaCl2 divide cells in 2 big Petri dishes add 20 ml 0.24 M CaCl2 and 15 ml Enzym Solution onto the cells incubate the cells by shaking them carefully for 3-4 h at room temperature transfer cells to 50 ml Greiner tubules zentrifuge 5 min at 1200 rpm decant supernatant add 30 ml CaCl2 and resuspend pellet by gentle shaking zentrifuge 5 min at 1200 rpm decant supernatant add 10 ml B5-Sucrose solution transfer cells to 15 ml Greiner tubules zentrifuge 5 min at 800 rpm remove the upper layer of swimming protoplasts with a cut pipette tip Counting of protoplasts create a 1:100 dilution (990 μl CaCl2 + 10 μl protoplasts) pipette 10 μl of the dilution onto the counting chamber count 4×5 group squares and determine the average cell number / μl = average × 50 × dilution Needed concentration for transfection: ca. 10 Mio/ml Transfection Mix 100 μl protoplasts (ca. 10 Mio/ml) + 5-10 μg plasmid (in a volume as little as possible) + 300 μl PEG invert the transfection reaction and incubate 10 min at room temperature drop by drop add 1 ml 0.275 M Ca(NO3)2 pH 6.0 add another 4 ml 0.275 M Ca(NO3)2 zentrifuge 5 min at 1200 rpm decant supernatant add 4 ml B5-Sucrose solution gently shake at room temperatur in the dark and over night add 5 ml 0.24 M CaCl2 centrifuge 10 min at 2500 rpm decant supernatant resuspend pellet in residuary liquid Evaluation occurs via Fluorescenc Microscope or via confocal Laser Microscope Solutions 0,275 M Ca(NO3)2 pH 6,0: 64,94 g/L Ca(NO3)2 0,39 g/l MES (2 mM) → adjust pH with KOH → autoclave PEG (250 ml): 62,5g PEG6000 (25%) 5,9 g Ca(NO3)2 (100 mM) 20,5 g Mannitol (450mM) → adjust pH 9,0 (Tris or Bicine for buffering) → filter sterilize 0,24 M CaCl2: 35,28 g/l CaCl2 → autoclave B5-Sucrose: Gamborg B5-Medium (Sigma, 1 Pack) 1 mg/l 2,4-D 97 g/l Saccharose (0,28 M) → adjust pH 5,5 with NaOH → filter sterililize Enzyme solution (30ml CaCl2): 0,06 g Macerozyme 0,2 g Cellulase → shaking incubation for 2-4 h at RT or o.N. at 4°C → filter sterililize