PEG LiAc ssDNA - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki
The standard transformation protocol use a transformation solution with this composition:
Reagents:
| Component | Volume (µL) |
|---|---|
| PEG 3500 50% w/v | 240 |
| LiAc 1.0 M | 36 |
| Boiled ss-carrier DNA | 50 |
| Plasmid DNA plus Water | 34 |
| Total | 360 |
We make a similar solution we call PLS (PEG-LiAc-ssDNA) using 4 M LiAc instead of 1M. This means we can add more DNA if needed. This solution specifies one more µL of ss-carrier (51 instead of 50). This was done to make the volume added to each transformation an even 300 µL.
| Component | Volume (µL) |
|---|---|
| PEG 3500 50% w/v | 240 |
| LiAc 4.0 M | 9 |
| Boiled ss-carrier DNA | 51 |
| Total | 300 |
To make 30 mL PLS:
| Component | Volume (µL) |
|---|---|
| PEG 3500 50% w/v | 24 mL or 25.88 g |
| LiAc 4.0 M | 900 µL |
| Boiled SS-carrier DNA | 5100 µL |
| total | 30000 µL |
| Density of 50% PEG solution is 1.07842 g/mL |
Add the PEG to a sterile 50 mL FALCON tube. Add the LiAc. Cool the tube on ice. Boil enough tubes of ssDNA. Cool the ssDNA tubes on ice. Add the boiled ssDNA to the PEG. Mix by vortexing and return the tube to ice ASAP. Let the bubbles disappear. Aliquot 1-2 mL in Eppendorf tubes. The aliquots are kept frozen at -20 in LGM4 "Elsa".
Usage:
- Add 300 µL to 1E8 washed and pelleted cells
- Add 60 µL DNA + water
- Proceed with resuspension and heat shock as per the original protocol.