Template:SBB-Protocols Mini2

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Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit

MINIPREP Procedure for Plasmid DNA Purification

  • Pellet the cells in 96 well block
  1. spin 5 min at 5500 (use 1 mL of culture if cells were grown in 2YT)
  2. flick supernatant out into sink
  3. If cells were grown in LB, pellet another mL of culture before proceeding.
  • Do an alkaline lysis
  1. Use multichannel to add 250uL of Quiagen Buffer P1 into each well. Resuspend the cells using the vortexer.
  2. Add 250uL of Quiagen Buffer P2 (a base that denatures everything and causes cells to lyse). Mix using vortexer Solution should become clearer and blue.
  3. Add 350uL of Quiagen Buffer N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Mix using the vortexer, make sure all of the blue has disappeared.
  4. Spin in centrifuge at 5500 for 10 minutes.
  5. Set up column strips using aluminum holder and 24-well block (to catch flow through).
  • Purify DNA on a column
  1. Carefully pipette liquid into columns, if you pipette up cell junk, then re-spin the block at 5500 for 5 min.
  2. Spin in centrifuge at 2500 for 2 min to pull the liquid through the columns.
  3. Dump liquid out of the 24-well block (the DNA should be stuck to the white resin)
  4. Wash each column by adding 500 uL of Quiagen Buffer PB.
  5. Spin in centrifuge at 2500 for 2 min, then flick out the liquid again.
  6. Wash with 750uL of Quiagen Buffer PE (washes the salts off the resins).
  7. Spin in centrifuge at 2500 for 2 min, then flick out liquid again.
  8. Spin in centrifuge at 5500 for 5 min to dry off all water and ethanol.
  9. Label new 96-well PCR plate and put columns in them.
  10. Elute DNA by squirting 100uL of water down the middle of the column (don't let it stick to the sides).
  11. Spin in centrifuge at 5500 for 3 min. (if the elution volume is very low, you can spin again)
  12. Take out columns and cover the plate.
  13. Clean up - note the A1 buffer is stored at 4degC and all the rest at room temperature.