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Purifying DNA from an enzymatic reaction
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Revision as of 12:06, 19 September 2008 by Riachalder (Talk | contribs)
To purify DNA from an enzymatic reaction we used the GE Healthcare illustra GFX PCR DNA and Gel Band Purification Kit, and the method outlined below is taken from their protocol manual included with the kit.
This can be found here:[1]
1) Sample Capture
- Add 500 μl Capture buffer type 2 to up to 100 μl sample.
Note: If sample volume is greater than 100 μl, divide the sample and purify using more than one GFX MicroSpin column.
- Mix thoroughly.
Note: If sample contains DNA greater than 5 kbp, do not vortex, as this may cause shearing of the DNA.
- For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.
2) Sample Binding
- Centrifuge Capture buffer type 2-sample mix briefly to collect the liquid at the bottom of the tube.
- Load the Capture buffer type 2-sample mix onto the assembled GFX MicroSpin column and Collection tube.
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the flow through by emptying theCollection tube. Place the GFX MicroSpin column back inside the Collection tube.
3) Wash & Dry
- Add 500 μl Wash buffer type 1 to the GFX MicroSpin column.
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube
4) Elution
- Add 10–50 μl Elution buffer type 4 OR type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
- Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
- Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.
- Proceed to downstream application. Store the purified DNA at -20°C.
