IGEM:UC Berkeley/2006/Minipreps

MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)

1. 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.
2. Place the tubes in a centrifuge in a symmetrical pattern and spin at top speed for 1 minute.
3. Flick media out into sink.
4. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
5. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
6. Add 350uL of N3 buffer (an acid of ~pH5 that causes all the junk the precipitate and leaves all the desired products in solution). Slowly invert a few times, then shake.
7. Spin in centrifuge at top speed for 10 minutes.
8. Label blue columns with a lab pen (not a sharpie because an ethanol wash would erase it)
9. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000rpm for 30 seconds.
10. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
11. Wash each column with 500 uL of PB buffer.
12. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
13. Wash with 750uL of PE buffer (washes the salts off the resins).
14. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
15. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
16. Label new tubes (include "Rescue" or "Mini") and put columns in them.
17. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
18. Spin in centrifuge at top speed for 30 seconds.
19. Take out columns and cap the tubes.
20. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

-SIL 6/8/06