Knight:PhoenIX Maxiprep Kit

Materials

 * PhoenIX™ Maxiprep Kit
 * Isopropanol
 * 70% Ethanol
 * Knight:5810R centrifuge

When using a new kit

 * 1) Assemble cardboard column rack.
 * 2) Note that the resuspension, lysis and neutralization buffer bottles are VERY hard to open.

Procedure
See official protocol. The steps below have been changed to accommodate our tubes and centrifuge.


 * 1) Place PhoenIX™ Maxi column in the assembled column rack.
 * 2) Place a beaker underneath to collect flow through.
 * 3) Add 30 ml of equilibration buffer (gray cap label) to the surface of the column and allow the liquid to drain by gravity flow.
 * 4) *Note: It will take 15-20 minutes for the column to drain completely.
 * 5) Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4&deg;C.
 * 6) Remove all traces of liquid medium from the bacterial cell pellet by pouring.
 * 7) *Trace media can affect subsequent steps.
 * 8) Add 10 ml of RNase A-containing cell resuspension buffer (yellow cap label) to the cell pellet and vortex until completely resuspended.
 * 9) *There is some resuspension buffer with RNase A added stored at 4&deg;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&deg;C on the door.
 * 10) Transfer resuspended cells to 50 mL conical tube.
 * 11) Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
 * 12) Mix thoroughly by inverting until the lysate appears to be homogeneous (5-6 inversions). DO NOT VORTEX.
 * 13) Incubate 5 minutes at room temperature.
 * 14) *Note: Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured.
 * 15) Add 10 ml of neutralization buffer (green cap label).
 * 16) Securely cap the tube and mix immediately by multiple inversions until a homogeneous suspension containing no viscous matter is obtained. DO NOT VORTEX.
 * 17) *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.
 * 18) Centrifuge at 9,000 x g for 20 mins at room temperature.
 * 19) *Note: The supernatant must at room temperature (18 – 25&deg;C) prior to loading on the column.
 * 20) Verify that the qquilibration buffer has been collected in the beaker.
 * 21) Discard the flow-through and replace the container.
 * 22) Use a pipette to remove the cleared lysate supernatant from the centrifuged sample and add to the top of the equilibrated column.
 * 23) *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.
 * 24) Allow the lysate to drain by gravity flow (10-15 minutes).
 * 25) Discard the flowthrough and replace the empty container.
 * 26) 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).
 * 27) 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).
 * 28) Discard the flow-through.
 * 29) Replace the waste collection container with a 50 mL conical tube.
 * 30) Add 15 ml of elution buffer (pink cap label) to the top of the column.
 * 31) Allow the eluate to drain by gravity flow (5-10 minutes) into the centrifuge tube.
 * 32) Add 10.5 ml of room temperature isopropanol to the eluted plasmid DNA in the centrifuge tube.
 * 33) Mix and centrifuge at 9,000 x g for 40 minutes at 4&deg;C.
 * 34) Pour out the supernatant taking care not to disturb the DNA pellet.
 * 35) Add 5 ml of room temperature 70% ethanol and wash the pellet.
 * 36) Centrifuge at 9,000 x g for 10 minutes at 4&deg;C.
 * 37) Completely remove ALL of the supernatant from the pellet with a pipette.
 * 38) Air-dry the pellet for 10 minutes.
 * 39) *Note: Drying with a vacuum chamber is not recommended because over-dried DNA may be difficult to completely resuspend.
 * 40) Dissolve the plasmid DNA in 500 &mu;l of water.
 * 41) Move to smaller tube.
 * 42) Take a spectrophotometer reading to assess concentration.