Protoplast Isolation and Transformation of an A. flavus pyr Mutant


  1. Start an overnight culture of 3357-5 by putting 1-3ml of Triton-X on an older, sporulating plate and scraping the whole plate using a glass spreader bar, add to 100ml of PDBU grow at: 200rpm and 28°C for 12-16hrs, 30°C 10-12h
  2. The following day freshly prepare the enzyme solution
  3. Pour the culture into 2 50ml conical tubes
  4. Centrifuge the overnight culture at “6” for 10 minutes in the clinical centrifuge. (Around 8k RPM in swinging bucket)
  5. Pour off supernatant and resuspend with sterile water.
  6. Centrifuge at “6” for 12 minutes in the clinical centrifuge.
  7. Pour off supernatant
  8. Filter the enzyme solution using a .45mm filter and add 15ml of the enzyme solution for each 5ml of tissue (each conical tube).
  9. Shake for 3-4hrs at 70rpm at 30-32°C
  10. Centrifuge at 200rpm for 1 minute in a swinging bucket rotor or at “2” for 1 minute in the clinical centrifuge.
  11. Pour the supernatant into a new 50ml conical tube and top off with 50ml of STC buffer, (I prefer to use a 50ml serological pipet to remove the supernatant).
  12. Centrifuge at 3000rpm for 5 minutes in a swinging bucket rotor or at “4” for 10 minutes in a clinical centrifuge
  13. Pour off supernatant
  14. Resuspend in 50ml STC buffer
  15. Repeat step #12
  16. Pour off supernatant leaving just a little STC in the bottom of each conical, be careful not to tilt the tube back up to soon or you’ll need to resuspend in STC and spin again.
  17. Create 1:10 and 1:20 dilutions in STC
  18. Using the hemocytometer, count the protoplasts present in five of the boxes
  19. Calculate total number of protoplasts using the formula:                                             protoplasts x dilution x 50,000
  20. If necessary dilute the protoplasts to1x108/ml, generally I find my concentration to be about 1×107/ml
  21. Add 100ml of diluted protoplasts to 1.5ml tubes (one with just protoplasts, one with just auxotrophic marker, and the rest with your plasmids)
  22. Add 1-10mg of each DNA to be transformed
  23. Mix by gently pipeting up and down
  24. Incubate on ice for 20 minutes
  25. Add 1ml of 50% PEG
  26. Mix by gently pipeting up and down
  27. Incubate at RT for 20 minutes
  28. Plate 200-300ml of protoplasts on the surface of recovery media
    1. MLS+U
  1. Protoplasts (negative control)
    1. MLS
  1. Protoplasts (positive control)
  1. Stack the plates and incubate at 37°C for 2-4 days.
  2. Pick colonies using sterile toothpick and transfer to 60mm PDB agar plates, grow 3-4days and isolate DNA using CTAB method if needed to pcr screen for knockout.


Materials and Solutions

PDBU (100ml)

  • 2.4g                 Potato Dextrose Broth
  • 0.112g             uracil
  • 100ml              diH2O


Enzyme Solution (30ml to be divided into 2)

  • 25.5 ml            1.2MNaCl (7g in 100ml diH2O)
  • 3ml                 0.2M NaPO4
  • 0.14g               Lysing enzyme (4°C)                                     Sigma #L-14122
  • 0.08g               Driselase (-20°C)                                            Sigma #D-9515
  • 300ml               b-glucorinidase (4°C)                                      Sigma #G-0876
  • 0.08g               BSA (4°C dry)                                                Sigma #A2153-100G


50% PEG (100ml)

  • 50g                  PEG (MW 4,000)
  • 1ml                  1M stock TrisCl (pH7.5)
  • 1ml                  1M stock CaCl2
  • Filter sterilize


STC Buffer (500ml)

  • 109.32g           Sorbitol
  • 5ml                  1M stock TrisCl (pH7.5)
  • 5ml                  1M stock CaCl2
  • 500ml              diH2O
  • Autoclave and store at 4°C


MLS (500ml)

  • 17.5g               Czapeck’s broth
  • 26.43g             0.4MNH4SO4
  • 5g                    agar


MLS+U (500ml)

  • 17.5g               Czapeck’s broth
  • 26.43g             0.4MNH4SO4
  • 5g                    agar
  • 0.561g             uracil