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


 * 1) zentrifuge 5 min at 800 rpm
 * 2) carefully decant supernatant
 * 3) resuspend pellet in 30 ml 0.24 M CaCl2
 * 4) divide cells in 2 big Petri dishes
 * 5) add 20 ml 0.24 M CaCl2 and 15 ml Enzym Solution onto the cells
 * 6) incubate the cells by shaking them carefully for 3-4 h at room temperature
 * 7) transfer cells to 50 ml Greiner tubules
 * 8) zentrifuge 5 min at 1200 rpm
 * 9) decant supernatant
 * 10) add 30 ml CaCl2 and resuspend pellet by gentle shaking
 * 11) zentrifuge 5 min at 1200 rpm
 * 12) decant supernatant
 * 13) add 10 ml B5-Sucrose solution
 * 14) transfer cells to 15 ml Greiner tubules
 * 15) zentrifuge 5 min at 800 rpm
 * 16) remove the upper layer of swimming protoplasts with a cut pipette tip

Counting of protoplasts


 * 1) create a 1:100 dilution (990 μl CaCl2 + 10 μl protoplasts)
 * 2) pipette 10 μl of the dilution onto the counting chamber
 * 3) count 4×5 group squares and determine the average
 * 4) cell number / μl = average × 50 × dilution
 * 5) Needed concentration for transfection: ca. 10 Mio/ml

Transfection


 * 1) Mix 100 μl protoplasts (ca. 10 Mio/ml) + 5-10 μg plasmid (in a volume as little as possible) + 300 μl PEG
 * 2) invert the transfection reaction and incubate 10 min at room temperature
 * 3) drop by drop add 1 ml 0.275 M Ca(NO3)2 pH 6.0
 * 4) add another 4 ml 0.275 M Ca(NO3)2
 * 5) zentrifuge 5 min at 1200 rpm
 * 6) decant supernatant
 * 7) add 4 ml B5-Sucrose solution
 * 8) gently shake at room temperatur in the dark and over night
 * 9) add 5 ml 0.24 M CaCl2
 * 10) centrifuge 10 min at 2500 rpm
 * 11) decant supernatant
 * 12) resuspend pellet in residuary liquid
 * 13) Evaluation occurs via Fluorescenc Microscope or via confocal Laser Microscope

Solutions

→ adjust pH with KOH → autoclave
 * 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 9,0 (Tris or Bicine for buffering) → filter sterilize
 * PEG (250 ml):
 * 62,5g PEG6000 (25%)
 * 5,9 g Ca(NO3)2 (100 mM)
 * 20,5 g Mannitol (450mM)

→ autoclave
 * 0,24 M CaCl2:
 * 35,28 g/l CaCl2

→ adjust pH 5,5 with NaOH → filter sterililize
 * B5-Sucrose:
 * Gamborg B5-Medium (Sigma, 1 Pack)
 * 1 mg/l 2,4-D
 * 97 g/l Saccharose (0,28 M)

→ shaking incubation for 2-4 h at RT or o.N. at 4°C → filter sterililize
 * Enzyme solution (30ml CaCl2):
 * 0,06 g Macerozyme
 * 0,2 g Cellulase