Qiagen-tip 100 Midiprep or Qiagen-tip 500 Maxiprep

 

Materials:

Note: amounts denoted by x/y are for QIAGEN-tip 100 / QIAGEN-tip 500.

Methods:

1. Chill Buffer P3 on ice (also P1?).
2. For each bacterial strain, use appropriate amount of culture according to above table.
3. Harvest the bacterial cells by centrifugation at 6000 g for 15 min . Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.
4. Resuspend the bacterial pellet in 4/10 ml of buffer P1. Vortex until there are no more clumps. Pipet the contents of the third tube into each of the other two tubes.
5. Add 4/10 ml of Buffer P2, mix gently but thoroughly by inverting 4-6 times, and incubate at room temprerature for 5 min. Do not vortex. Do not allow lysis reaction to proceed for more than 5 min. Close Buffer P2 bottle immediately.
6. Add 4/10 ml of chilled Buffer P3, mix immediately but gently by inverting 4-6 times, and incubate on ice for 15/20 min.
7. Mix the sample again, before loading the centrifuge. Centrifuge at for 30 min . Remove supernatant containing plasmid DNA promptly. After centrifugation, the supernatant should be clear.
8. Re-centrifuge the supernatant at for 15 min . Remove supernatant containing plasmid DNA promptly.
9. Equilibrate a QIAGEN-tip 100/500 by applying 4/10 ml Buffer QBT, and allow the column to empty by gravity flow.
10. Apply the supernatant of plasmid DNA to the QIAGEN-tip and allow it to enter the resin by gravity flow. The supernatant should be loaded onto the QIAGEN-tip promptly. If it becomes cloudy, it must be recentrifuged.
11. Wash the QIAGEN-tip with ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
12. Elute DNA with 5/15 ml Buffer QF. Collect the eluate in a 10 ml or 30 ml tube. Do not use polycarbonate tubes because they are not resistant to the alcohol used in subsequent steps.
13. Precipitate DNA by adding 3.5/10.5 (0.7 volumes) room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at for 30 min . Carefully decant the supernatant. Mark the outside of the tube before centrifugation to locate pellet more easily.
14. Wash DNA pellet with 2/5 ml of room temperature ethanol, and centrifuge at for 10 min. Carefully decant the supernatant without disturbing the pellet.
15. Air-dry the pellet for 5-10 min, and redissolve the DNA in a suitable volume of buffer (e.g. TE, pH 8.0, or 10 mM Tris-Cl, pH 8.5). Redissolve the DNA pellet by rinsing the walls to recover all the DNA. Do not pipet the DNA up and down, to avoid sheering.