Griffitts:PCR clean-up: Difference between revisions
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==Procedure== | ==Procedure== | ||
* Label columns and microcentrifuge tubes with the names of the appropriate PCR reactions | * Label spin columns and microcentrifuge tubes with the names of the appropriate PCR reactions | ||
* Using P-200, add 200 μL buffer PB to each 40-μL PCR and mix well by gently | * Using a P-200, add 200 μL buffer PB to each 40-μL PCR and mix well by gently pipetting up and down | ||
* Transfer mixture to a QiaPrep spin column | * Transfer the mixture to a QiaPrep spin column | ||
* Centrifuge at full speed for 30 seconds | * Centrifuge at full speed for 30 seconds | ||
* Dump and tap | * Dump and tap | ||
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* Centrifuge at full speed for 30 seconds | * Centrifuge at full speed for 30 seconds | ||
* Dump and tap | * Dump and tap | ||
* Centrifuge for an additional | * Centrifuge for an additional 5 minutes at full speed | ||
* Move column to a 1.5 mL microfuge tube | * Move the column to a 1.5 mL microfuge tube | ||
* Add 50 μL | * Add 50 μL buffer EB (to elute) | ||
** Drip this directly onto the filter disk but don't touch it with the pipette tip | ** Drip this directly onto the filter disk but don't touch it with the pipette tip | ||
* Wait | * Wait 2 minutes | ||
* Centrifuge at full speed for 1 minute | * Centrifuge at full speed for 1 minute | ||
** Point the lids of the microcentrifuge tubes in a clockwise direction, otherwise they'll break off | ** Point the lids of the microcentrifuge tubes in a clockwise direction, otherwise they'll break off | ||
Revision as of 12:24, 30 September 2010
Materials
- Buffer PB (in Qiagen kit)
- Buffer PE (in Qiagen kit)
- Buffer EB (in Qiagen kit)
- QiaPrep spin column (1 per PCR reaction)
- 1.5 mL microcentrifuge tube (1 per PCR reaction)
Procedure
- Label spin columns and microcentrifuge tubes with the names of the appropriate PCR reactions
- Using a P-200, add 200 μL buffer PB to each 40-μL PCR and mix well by gently pipetting up and down
- Transfer the mixture to a QiaPrep spin column
- Centrifuge at full speed for 30 seconds
- Dump and tap
- Add 600 μL buffer PE to each column
- Centrifuge at full speed for 30 seconds
- Dump and tap
- Centrifuge for an additional 5 minutes at full speed
- Move the column to a 1.5 mL microfuge tube
- Add 50 μL buffer EB (to elute)
- Drip this directly onto the filter disk but don't touch it with the pipette tip
- Wait 2 minutes
- Centrifuge at full speed for 1 minute
- Point the lids of the microcentrifuge tubes in a clockwise direction, otherwise they'll break off
