Protocol Code: CT5

Protoplast Transformation

Solutions needed

• SMMP + lysozyme
• TE buffer
• 2x SMM
• PEG solution
• DM3 plates
• Antibiotic plates

Procedure

1. Centrifuge cells and suspend in 1/10 volume of SMMP solution
2. Add lysozyme @ 2mg/ml
3. Incubate @ 37˚C for 2h
4. Pellet cells at 2600g for 15 min
5. Wash once with SMMP and pellet again
6. Bring to 1/10-1/15 volume of starting culture by adding SMMP
7. This is stable for ~ 5h at room temp
8. Add 5ug or less DNA to 50ul TE buffer and mix with equal volume of 2x SMM solution
9. Add 0.5ml protoplast solution
10. Immediately add 1.5ml of 40%(w/v) PEG
11. After 2 min add 5 ml SMMP medium
12. Centrifuge for 10 min at 2600g
13. Resuspend in 1ml SMMP and incubate for 2-3h at 37˚C
14. Plate on DM3 recovery plates for a day (can try going straight to antibiotic)
15. Plate colonies on antibiotic plates