"
Team:EPF-Lausanne/Protocols/Miniprep
From 2011.igem.org
Miniprep
The miniprep is used to extract plasmids from a bacterial colony. The bacteria cells are lysed to release the plasmids, then the plasmids are filtered out, and finally their concentration is measured.
TODO: could somebody please provide some details on how the colonies are made?
Contents |
Equipment
- Invitrogen PureLink Quick miniprep kit: white and red box, kept in the storage room.
- Resuspension Buffer: kept in the fridge. It's part of the miniprep kit, but kept cool since it contains RNAase.
- Centrifuge
- Nanodrop 1000 Spectrophotometer: on the left on the computer at the entrance of Sebastian's lab.
Notes
- As of 8 June 2011, all the solutions in the kit are already mixed. Therefore, most of the "Before Starting" steps of the instruction leaflet can be skipped.
- The TE buffer is replaced by EB buffer. The TE buffer inhibits sequencing reactions, therefore it is avoided.
Protocol
Miniprep
All the steps of the miniprep are described in the instruction leaflet that is included in the miniprep kit. Just heed these extra warnings:
- To "resuspend", wash the solution up and down in a micropipette. Be careful to not create any foam.
- Return the resuspension buffer to the refrigerator once it is no longer used.
Concentration measurement
Plasmid concentration is finally measured using the Nanodrop 1000 photospectrometer. It is controlled by the "ND-1000" software (icon on the desktop).
- Calibrate the spectrometer: deposit a 1 ul drop of buffer solution onto the sensor, and click the "blank" button.
- Measure plamsid concentration: deposit 1 ul of plasmid solution onto the sensor, and measure with the "measure" button. The reported plasmid concentration is considered valid if the "260/280" ratio is larger than 1.80.
