"
User:Jn323/1 August 2012
From 2012.igem.org
Contents |
Electrophoresis
Preparation of Cells
- Check O/N growth
- OD600 reading - 1.8
- Back-Dilution prepared until OD600 reading was between 0.4 and 0.8
Following steps done on ice
- Place following items on ice (for future use):
- ultra pure dH2O (DNase, RNase free)
- 0.1cm cuvettes
- DNA sample
- Aliquot 1mL of grown cells into 1.5mL eppendorf tubes each
- Wash cells twice in 1x volume of cold ultra pure distilled water
- Spin down each aliquot in microcentrifuge (4000rpm, 10 minutes at 4°C)
- Remove supernatant immediately
- Repeat steps a & b
- Spin down aliquots once more in same conditions
- Resuspend each pellet in 50uL of cold ultra-pure distilled water with 50ng of DNA sample
- Transfer each aliquot into 0.1cm cuvettes
Electroporation
- Wipe sides of cuvettes prior to electroporation
- Electroporate at following settings:
- 1.8kV
- 200Ω
- 25 uF
- Immediately add 1mL SOC media
- Incubate cultures in shaking incubator at 30°C for 50 minutes
- Transfer SOC-cell suspension to clean 1.5mL eppendorf tubes
- Spin down in table-top centrifuge for 30 seconds
- Remove 950uL of supernatant to concentrate in 50uL of LB
- Resuspend and plate with either autoclaved beads
Results
Electroporation Results
| Cell Strain | TC (ms) | V (V) |
|---|---|---|
| ADP1 | 5.0 | 1788 |
| ADP1 - Control | 5.2 | 1788 |
| EcNR2 | 5.2 | 1789 |
| EcNR2 - Control | 5.3 | 1788 |
| BS168 | 5.2 | 1788 |
| BS168 - Control | 5.3 | 1788 |
| BSPERM739 | 5.2 | 1788 |
| BSPERM739 - Control | 5.3 | 1788 |
| BSPY79 | 5.2 | 1788 |
| BSPY79 - Control | 5.3 | 1788 |
| BS1A833 | 5.1 | 1789 |
| BS1A833 - Control | 5.2 | 1788 |
Plating Results
Observations
ADP1 and EcNR2 grew in a lawn with a great quantity of small colonies interspersed within the lawn. For future experiments, the concentration of cells should be decreased. BSPY79 showed small spreads of colonies. BS1A833 and BS168 showed no growth. All of the above mentioned strains had no growth on the negative control plates. BSPERM739 showed lawns similar to ADP1 and EcNR2, but the negative control showed the same result. This is most likely due to the fact that BSPERM739 is naturally resistant to neomycin, which is almost identical to kanomycin resistance.
