Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit
MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification
- Pellet the cells (for individual cultures)
- Pellet around 1.5 mL to 3 mL saturated culture by spinning full speed, 30 seconds.
- Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL if desired)
- Pellet the cells (for 24-well blocks)
- In 327, weigh the block on the scale, fill a dirty block with water to the same weight
- Use the Allegra centrifuge to spin at 5500rpm for 4 minutes
- Dump supernatant
- You will need to transfer the cells to microcentrifuge tubes after resuspension in A1 in the alkaline lysis section below
- Do an alkaline lysis
- Add 250uL of P1 (A1) buffer into each tube. Resuspend the cells using a vortexer.
- Add 250uL of P2 (A2) buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
- Add 300uL of N3 (A3) 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). Slowly invert a few times, then shake.
- Spin in centrifuge at top speed for 5 minutes.
- Label columns with an alcohol-resistant lab pen.
- Purify DNA on a column
- Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
- Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
- Wash each column with 500 uL of PB (AW) buffer.
- Spin in centrifuge at full speed for approximately 15 seconds, then flick out the liquid again.
- Wash with 750uL of PE (A4) buffer (washes the salts off the resins).
- Spin in centrifuge at full speed for approximately 15 seconds and flick out liquid again.
- Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
- Label new tubes and put columns in them.
- Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
- Spin in centrifuge at top speed for 30 seconds.
- Take out columns and cap the tubes.
- Clean up - note the P1 (A1) buffer is stored at 4degC and all the rest at room temperature.