Team:Brown-Stanford/Lab/Protocols/ProtoplastTransform
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<html><h1>Protocol Code: CT5</h1></html> | <html><h1>Protocol Code: CT5</h1></html> | ||
== '''Protoplast Transformation''' == | == '''Protoplast Transformation''' == | ||
+ | ===Solutions needed=== | ||
+ | *SMMP + lysozyme | ||
+ | *TE buffer | ||
+ | *2x SMM | ||
+ | *PEG solution | ||
+ | *DM3 plates | ||
+ | *Antibiotic plates | ||
- | + | ===Procedure=== | |
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#Centrifuge cells and suspend in 1/10 volume of SMMP solution | #Centrifuge cells and suspend in 1/10 volume of SMMP solution | ||
#Add lysozyme @ 2mg/ml | #Add lysozyme @ 2mg/ml |
Latest revision as of 18:18, 28 September 2011
Protocol Code: CT5
Protoplast Transformation
Solutions needed
- SMMP + lysozyme
- TE buffer
- 2x SMM
- PEG solution
- DM3 plates
- Antibiotic plates
Procedure
- Centrifuge cells and suspend in 1/10 volume of SMMP solution
- Add lysozyme @ 2mg/ml
- Incubate @ 37˚C for 2h
- Pellet cells at 2600g for 15 min
- Wash once with SMMP and pellet again
- Bring to 1/10-1/15 volume of starting culture by adding SMMP
- This is stable for ~ 5h at room temp
- Add 5ug or less DNA to 50ul TE buffer and mix with equal volume of 2x SMM solution
- Add 0.5ml protoplast solution
- Immediately add 1.5ml of 40%(w/v) PEG
- After 2 min add 5 ml SMMP medium
- Centrifuge for 10 min at 2600g
- Resuspend in 1ml SMMP and incubate for 2-3h at 37˚C
- Plate on DM3 recovery plates for a day (can try going straight to antibiotic)
- Plate colonies on antibiotic plates