Knight:PhoenIX Maxiprep Kit

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(Procedure)
Current revision (20:06, 10 January 2007) (view source)
(Procedure)
 
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#*''Trace media can affect subsequent steps.''
#*''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.
#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.
+
#*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.
#Transfer resuspended cells to 50 mL conical tube.
#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.  
#Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.  

Current revision

Contents

Materials

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.
    • Note: It will take 15-20 minutes for the column to drain completely.
  4. Pellet 200 ml of bacterial culture by centrifugation at 3,000 x g for 20 minutes at 4°C.
  5. Remove all traces of liquid medium from the bacterial cell pellet by pouring.
    • Trace media can affect subsequent steps.
  6. 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.
  7. Transfer resuspended cells to 50 mL conical tube.
  8. Add 10 ml of Lysis Buffer (blue cap label) and securely cap the tube.
  9. Mix thoroughly by inverting until the lysate appears to be homogeneous (5-6 inversions). DO NOT VORTEX.
  10. Incubate 5 minutes at room temperature.
    • Note: Do not incubate for longer than 5 minutes or plasmid DNA might become irreversibly denatured.
  11. Add 10 ml of neutralization buffer (green cap label).
  12. 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.
  13. 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.
  14. Verify that the qquilibration buffer has been collected in the beaker.
  15. Discard the flow-through and replace the container.
  16. 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.
  17. Allow the lysate to drain by gravity flow (10-15 minutes).
  18. Discard the flowthrough and replace the empty container.
  19. 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).
  20. 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).
  21. Discard the flow-through.
  22. Replace the waste collection container with a 50 mL conical tube.
  23. Add 15 ml of elution buffer (pink cap label) to the top of the column.
  24. Allow the eluate to drain by gravity flow (5-10 minutes) into the centrifuge tube.
  25. Add 10.5 ml of room temperature isopropanol to the eluted plasmid DNA in the centrifuge tube.
  26. Mix and centrifuge at 9,000 x g for 40 minutes at 4°C.
  27. Pour out the supernatant taking care not to disturb the DNA pellet.
  28. Add 5 ml of room temperature 70% ethanol and wash the pellet.
  29. Centrifuge at 9,000 x g for 10 minutes at 4°C.
  30. Completely remove ALL of the supernatant from the pellet with a pipette.
  31. 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.
  32. Dissolve the plasmid DNA in 500 μl of water.
  33. Move to smaller tube.
  34. 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.
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