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

  • 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

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