MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
- Palette cells by centifuging from liquid culture. If the culture is on a plate, resuspend the bacteria in 1mL LB using a scraper and pipette into a tube. Dip the scraper in ethanol and burn it to kill cells before paletting another plate.
- Place the tubes in a centrifuge in a symmetrical pattern and spin at top speed for 1 minute.
- Flick media out into sink.
- Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
- Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
- Add 350uL of 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). Slowly invert a few times, then shake.
- Spin in centrifuge at top speed for 10 minutes.
- Label blue columns with an alcohol-resistant lab pen.
- Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 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 buffer.
- Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
- Wash with 750uL of PE buffer (washes the salts off the resins).
- Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
- Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
- Label new tubes (include "Rescue" or "Mini") 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 buffer is stored at 4degC and all the rest at room temperature.