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Team:EPF-Lausanne/Protocols/Miniprep
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Plasmid preparation procedure known as "miniprep" is used to extract plasmids from small bacterial cultures. Bacteria undergo alkaline lysis to release the plasmids, while genomic DNA stays bound to cell structures. Cell debris are eliminated by centrifugation and the supernatant containing plasmids is loaded to a DNA binding column, it is first washed, than DNA is eluted. DNA conentration can be measured with a spectrophotometer. | Plasmid preparation procedure known as "miniprep" is used to extract plasmids from small bacterial cultures. Bacteria undergo alkaline lysis to release the plasmids, while genomic DNA stays bound to cell structures. Cell debris are eliminated by centrifugation and the supernatant containing plasmids is loaded to a DNA binding column, it is first washed, than DNA is eluted. DNA conentration can be measured with a spectrophotometer. | ||
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Plasmid concentration is finally measured using the Nanodrop 1000 photospectrometer. It is controlled by the "ND-1000" software (icon on the desktop). | Plasmid concentration is finally measured using the Nanodrop 1000 photospectrometer. It is controlled by the "ND-1000" software (icon on the desktop). | ||
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| + | * '''Load nucleic acid program''': if necessary, click "exit" to return to the main menu, then "Nucleic Acid". Initialise as instructed, by depositing a ul drop of water on the sensor. | ||
* '''Calibrate the spectrometer''': deposit a 1 ul drop of buffer solution onto the sensor, and click the "blank" button. | * '''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. | * '''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. | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Latest revision as of 08:49, 15 July 2011
Plasmid preparation - Miniprep
Back to protocols.Plasmid preparation procedure known as "miniprep" is used to extract plasmids from small bacterial cultures. Bacteria undergo alkaline lysis to release the plasmids, while genomic DNA stays bound to cell structures. Cell debris are eliminated by centrifugation and the supernatant containing plasmids is loaded to a DNA binding column, it is first washed, than DNA is eluted. DNA conentration can be measured with a spectrophotometer.
For miniprep procedure bacterial cultures are grown overnight in 5ml LB with with antibiotic. These cultures are started from a single colony grown on a LB-agar plate, usually the selection plate after transformation or it can be a streak of glycerin stock.
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 with a rotor for Eppendorf tubes
- Nanodrop 1000 Spectrophotometer: on the left of 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. For steps with DNA binding column centrifuge is used, rather than vacuum. Just heed these extra warnings:
- To "resuspend", wash the solution up and down in a micropipette. Be careful to not create foam.
- Return the resuspension buffer to the refrigerator when it is not longer needed.
Concentration measurement
Plasmid concentration is finally measured using the Nanodrop 1000 photospectrometer. It is controlled by the "ND-1000" software (icon on the desktop).
- Load nucleic acid program: if necessary, click "exit" to return to the main menu, then "Nucleic Acid". Initialise as instructed, by depositing a ul drop of water on the sensor.
- 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.
