Arkin:JCAProtocols Miniprep

Protocols Page

[|How to do a Miniprep]

MINIPREP (1mL - 5mL) Procedure for Plasmid DNA Purification (using the QIAGEN QIAPrep Spin Miniprep kit)
 * 1) Pellet 1.5 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)
 * 3) Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
 * 4) Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
 * 5) 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.
 * 6) Spin in centrifuge at top speed for 5 minutes.
 * 7) Label blue columns with an alcohol-resistant lab pen.
 * 8) Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
 * 9) Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
 * 10) Wash each column with 500 uL of PB buffer.
 * 11) Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
 * 12) Wash with 750uL of PE buffer (washes the salts off the resins).
 * 13) Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
 * 14) Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
 * 15) Label new tubes and put columns in them.
 * 16) Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
 * 17) Spin in centrifuge at top speed for 30 seconds.
 * 18) Take out columns and cap the tubes.
 * 19) Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.