Template:SBB-Protocols Micro4

Miniprep purification of DNA using Macherey-Nagel Nucleospin Kit

MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification

• Pellet the cells (for individual cultures)

1. Pellet around 1.5 mL to 3 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL if desired)

• Pellet the cells (for 24-well blocks)

1. In 327, weigh the block on the scale, fill a dirty block with water to the same weight
2. Use the Allegra centrifuge to spin at 5500rpm for 4 minutes
3. Dump supernatant
4. You will need to transfer the cells to microcentrifuge tubes after resuspension in A1 in the alkaline lysis section below

• Do an alkaline lysis

1. Add 250uL of P1 (A1) buffer into each tube. Resuspend the cells using a vortexer.
2. 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.
3. 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.
4. Spin in centrifuge at top speed for 5 minutes.
5. Label columns with an alcohol-resistant lab pen.

• Purify DNA on a column

1. Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
2. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
3. Wash each column with 500 uL of PB (AW) buffer.
4. Spin in centrifuge at full speed for approximately 15 seconds, then flick out the liquid again.
5. Wash with 750uL of PE (A4) buffer (washes the salts off the resins).
6. Spin in centrifuge at full speed for approximately 15 seconds and flick out liquid again.
7. Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
8. Label new tubes and put columns in them.
9. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
10. Spin in centrifuge at top speed for 30 seconds.
11. Take out columns and cap the tubes.
12. Clean up - note the P1 (A1) buffer is stored at 4degC and all the rest at room temperature.