The standard transformation protocol use a transformation solution with this composition:
Reagents:
| Component | Volume (µL) | Final |
|---|---|---|
| PEG 3500 50% w/v | 240 | 33% |
| LiAc 1.0 M | 36 | 0.1M |
| 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.
PLS (PEG-LiAc-ssDNA)
| Component | Volume (µL) |
|---|---|
| PEG 3500 50% w/v | 240 |
| LiAc 4.0 M | 9 |
| Boiled ss-carrier DNA | 51 |
| Plasmid DNA plus Water | 60 |
| Total | 360 |
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
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.