NOTICE: You are viewing a page of the openwetware wiki. Our "dewikify" feature makes a wiki page appear as a normal web page. In April 2017, this feature will GO AWAY and this URL will redirect to the source URL on our wiki. We're sorry for the inconvenience.

home        research        people        papers        teaching        join us        internal        pictures        contact       


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