DNA Fragment Purification
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TE solution
- 10 mM Tris (pH to 7.5)
- 1 mM EDTA (pH to 8.0 to dissolve)
- Frozen agarose gel piece containing the desired DNA fragment
- Micropipetter and tips
- Microcentrifuge and tubes
- Spatula
- PCR machine and the 600-ul tubes for use in the machine
- Ultrafree-MC(R) filter units, 0.45 um
- Quick thaw the gel piece in a 600-ul tube using the PCR machine at 37 degrees C
- Macerate the gel piece with a spatula.
- Transfer into the sample cup of the filter unit.
- Spin the filter unit for 20 minutes at 5,000 g.
- Transfer fluid in the bottom of the filter unit to a new tube and place on ice.
- Add 200 ul of TE to the sample cup of the filter unit and incubate at room temperature for 10 minutes.
- Spin the filter unit for 20 minutes at 10,000 g.
- Combine with the first collection and estimate the total volume.
- Add 0.1 volume of the sodium acetate and 3 volumes of 100 % ethanol.
- Store the tubes at -20 degrees C for 1 hour.
- Spin in a microcentrifuge at maximum speed for 15 minutes.
- Discard supernatant and add 400 ul of 70 % ethanol.
- Spin in a microcentrifuge at maximum speed for 5 minutes.
- Discard the ethanol and add another 400 ul of 70 % ethanol.
- Spin in a microcentrifuge at maximum speed for 5 minutes.
- Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
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Resuspend the pellet in 20 ul of distilled water and keep at 4 degrees C or -20 degrees C for storage and for estimation of DNA concentration (LAB 4) later.
- This method of DNA fragment purification works well with fragment sizes less than 5,000 basepairs. The yield, based on subsequent estimation of DNA concentration (LAB 4), is typically 50% or better depending on the size.
![HA/HA蛋白Recombinant Influenza A virus Hemagglutinin (HA)重组蛋白HA; Hemagglutinin [Cleaved into: Hemagglutinin HA1 chain]; Fragment蛋白](https://img1.dxycdn.com/p/s14/2024/0914/429/2534610676379384381.jpg!wh200)
