IGEM:IMPERIAL/2009/Transforming Competent Cells: Difference between revisions
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*Electrocuvettes | *Electrocuvettes | ||
*Eppendorf tubes | *Eppendorf tubes | ||
*P1000 and P200 Gilsons | *P1000 and P200 Gilsons and tips | ||
===Reagents=== | ===Reagents=== | ||
Latest revision as of 07:35, 12 August 2009
Aims
To transform our electrocompetent E. coli with plasmids containing selected DNA/Parts/BioBricks.
Equipment
- Desktop centrifuge
- Electroporation machine
- Electrocuvettes
- Eppendorf tubes
- P1000 and P200 Gilsons and tips
Reagents
- XL-1 Blue competent cells (stored in the freezer)
- LB
- Plates with required antibiotic
Protocol
- 1ul DNA into competent cell suspension. Pipette up and down.
- Take 50ul – put into electro-cuvette. Tap to ensure even distribution.
- Electroporate – make sure the machine is set to ‘bacteria’ on the mV setting. You should get a reading between 3.00 and 4.00 on the m/s read-out.
- Add 450ul LB to cuvette. Pipette up and down.
- Take 450ul and put into a new eppendorf tube. Put in the shaking incubator for 30mins.
- Centrifuge for 1minute.
- Discard supernatant.
- Plate out cells.
- Incubate overnight at 37°C
