Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit
MINIPREP Procedure for Plasmid DNA Purification
- Pellet the cells in 96 well block
- spin 5 min at 5500 (use 1 mL of culture if cells were grown in 2YT)
- flick supernatant out into sink
- If cells were grown in LB, pellet another mL of culture before proceeding.
- Do an alkaline lysis
- Use multichannel to add 250uL of Quiagen Buffer P1 into each well. Resuspend the cells using the vortexer.
- 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.
- 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.
- Spin in centrifuge at 5500 for 10 minutes.
- Set up column strips using aluminum holder and 24-well block (to catch flow through).
- Purify DNA on a column
- Carefully pipette liquid into columns, if you pipette up cell junk, then re-spin the block at 5500 for 5 min.
- Spin in centrifuge at 2500 for 2 min to pull the liquid through the columns.
- Dump liquid out of the 24-well block (the DNA should be stuck to the white resin)
- Wash each column by adding 500 uL of Quiagen Buffer PB.
- Spin in centrifuge at 2500 for 2 min, then flick out the liquid again.
- Wash with 750uL of Quiagen Buffer PE (washes the salts off the resins).
- Spin in centrifuge at 2500 for 2 min, then flick out liquid again.
- Spin in centrifuge at 5500 for 5 min to dry off all water and ethanol.
- Label new 96-well PCR plate and put columns in them.
- Elute DNA by squirting 100uL of water down the middle of the column (don't let it stick to the sides).
- Spin in centrifuge at 5500 for 3 min. (if the elution volume is very low, you can spin again)
- Take out columns and cover the plate.
- Clean up - note the A1 buffer is stored at 4degC and all the rest at room temperature.