The Plasmid Minipred Kit could obtain 5-30 μg pure plasmids DNA from 1-4 ml culture. And the ratio of OD260/OD280 is generally between 1.8 and 2.0. Plasmid DNA prepared using the Plasmid Minipred Kit is suitable for a variety of routine applications including PCR, enzyme digestion, sequencing, in vitro transcription, ligation and transformation.
Standard DNA Purification Protocol
1. Make sure that Solution A and Buffer W2 have been prepared as described above.
2. Pellet 1-4 mL bacterial cells cultured overnight, and centrifuge at 12,000 rpm for 30 seconds and discard supernatant .(Remove excess media possibly.)
3. Resuspend the cell pellets in 250 μL Solution A (RNase A added). No cell clumps should be visible after resuspension of the pellet.
4. Add 250 μL Solution B and mix gently by inverting the tube 4-6 times. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Limit the lysis reaction time within 5 minutes.
5. Add 350 μL Buffer N and mix immediately and thoroughly by inverting tube 4-6 times. (To avoid localized precipitation, mix the solution thoroughly, immediately after adding Buffer N. )
6. Centrifuge for 10 minutes at 12,000 rpm, and keep the supernatant. Place the Spin Column into Collection Tube, and add the supernatant to Spin Column.
7. Centrifuge for 1 minute at 12,000 rpm and discard the flow-through.
8. Wash the Spin Column by adding 500 μL Buffer W1 and centrifuging for 1 minute at 12,000 rpm. Discard the flow-through.
9. Wash the Spin Column by adding 700 μL Buffer W2 and centrifuge for 1 minute at 12,000 rpm. Discard the flow-through.
10. Wash the Spin Column by adding additional 500 μL Buffer W2 and centrifuge for 1 minute at 12,000 rpm. Discard the flow-through.
11. Centrifuge at 12,000 rpm for additional 1 minute to remove residual washing buffer. (Residual washing buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer W2 may inhibit subsequent enzymatic reactions.)
12. Place the Spin Column in a clean 1.5 mL microcentrifuge tube (not provided) and add 50-80 μL Elution Buffer* (or TE PH 8.5 or water ) to the center of the membrane. Incubate for 1-2 minute at room temperature and centrifuge 12,000 rpm for 1 minute.
*The volume of Elution Buffer cannot be less than 50 μL.
DNA recovery can be increased by using pre-warmed Elution Buffer at 60 °C.