When using a new kit
- Assemble the cardboard column rack.
- Note that the resuspension, lysis, and neutralization buffer bottles are VERY hard to open.
Procedure
The steps below have been changed to accommodate our tubes and centrifuge.
- Place the PhoenIXâ„¢ Maxi column in the assembled column rack.
- Place a beaker underneath to collect flow through.
- Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to drain by gravity flow.
- Note: It will take 15-20 minutes for the column to drain completely.
- Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.
- Remove all traces of liquid medium from the bacterial cell pellet by pouring.
- Trace media can affect subsequent steps.
- Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
- There is some resuspension buffer with RNase A added stored at 4°C in 32-306. To make more, you’ll need to add RNase A to more resuspension buffer. The resuspension buffer (without RNase A) is in the box and the RNase A is stored at -20°C on the door.
- Transfer resuspended cells to 50 mL conical tube.
- Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
- Mix thoroughly by inverting until the lysate appears to be homogeneous (5-6 inversions). DO NOT VORTEX.
- Incubate 5 minutes at room temperature.
- Note: Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured.
- Add 10 ml of neutralization buffer (green cap label).
- Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.
- Note: If preparing several samples at once, thoroughly mix each sample immediately after the addition of the neutralization buffer before adding the buffer to the next tube.
- Centrifuge at 9,000 x g for 20 mins at room temperature.
- Note: The supernatant must at room temperature (18 – 25°C) prior to loading on the column.
- Verify that the qquilibration buffer has been collected in the beaker.
- Discard the flow-through and replace the container.
- Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.
- Note: Do not pour lysate directly onto the column. Use a pipette to ensure that precipitate particles do not enter the column and cause clogging.
- Allow the lysate to drain by gravity flow (10-15 minutes).
- Discard the flowthrough and replace the empty container.
- Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
- Add 30 ml of column wash buffer (orange cap label) to the top of the column and allow the liquid to drain by gravity flow (10 minutes).
- Discard the flow-through.
- Replace the waste collection container with a 50 mL conical tube.
- Add 15 ml of elution buffer (pink cap label) to the top of the column.
- Allow the eluate to drain by gravity flow (5-10 minutes) into the centrifuge tube.
- Add 10.5 ml of room temperature isopropanol to the eluted plasmid DNA in the centrifuge tube.
- Mix and centrifuge at 9,000 x g for 40 minutes at 4°C.
- Pour out the supernatant taking care not to disturb the DNA pellet.
- Add 5 ml of room temperature 70% ethanol and wash the pellet.
- Centrifuge at 9,000 x g for 10 minutes at 4°C.
- Completely remove ALL of the supernatant from the pellet with a pipette.
- Air-dry the pellet for 10 minutes.
- Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.
- Dissolve the plasmid DNA in 500 μl of water.
- Move to smaller tube.
- Take a spectrophotometer reading to assess concentration.
Notes
- These spin steps may not be hard enough. Most of the purification steps are supposed to be at 12,000 x g.
- You’re not supposed to let the column stand between steps.