Team:Calgary/Notebook/Protocols/PCRPurification
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<li>Discard liquid and repeat last step with 500µl of DNA Wash Buffer </li> | <li>Discard liquid and repeat last step with 500µl of DNA Wash Buffer </li> | ||
<li>Discard liquid and centrifuge the empty HiBind DNA column for 2 min at maximum speed to dry the column matrix</li> | <li>Discard liquid and centrifuge the empty HiBind DNA column for 2 min at maximum speed to dry the column matrix</li> | ||
- | <li>Transfer HiBind DNA column to a clean 1.5mL tube and elute with 30-50µL of | + | <li>Transfer HiBind DNA column to a clean 1.5mL tube and elute with 30-50µL of ddH<sub>2</sub>O</li> |
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li> | <li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li> | ||
</ol> | </ol> |
Latest revision as of 00:20, 28 September 2013
PCR Purification
PCR Purification
For PCR purification, we used the E.Z.N.A.™ Cycle-Pure Kit. This kit is used to purify PCR products, as well DNA products in enzymatic reactions, particularly, restriction digests. We followed the manufacturer’s Spin Protocol.
Reagents and Materials
- E.Z.N.A.™ Cycle-Pure Kit
- PCR products
Protocol
- Determine the volume of the PCR reaction, transfer the sample into a clean 1.5ml microcentrifuge tube, and add 4-5 volumes of Buffer CP
- Vortex thoroughly to mix
- Spin down tubes shortly
- Place a HiBind DNA column in a provided 2ml collection tube
- Add 100µl Equilibration Buffer to the column
- Incubate at room temperature for 4 minutes
- Spin at maximum speed for 20 seconds
- Apply the sample to the HiBind DNA column and centrifuge at 10,000x g for 1 min at room temperature
- Discard liquid
- Wash the HiBind DNA column by adding 700µl of DNA Wash Buffer diluted with absolute ethanol and centrifuge centrifuge at 10,000x g for 1 min at room temperature
- Discard liquid and repeat last step with 500µl of DNA Wash Buffer
- Discard liquid and centrifuge the empty HiBind DNA column for 2 min at maximum speed to dry the column matrix
- Transfer HiBind DNA column to a clean 1.5mL tube and elute with 30-50µL of ddH2O
- Use a spectrophotometer to measure the concentration and the purity of your plasmid