3.Add 0.3 ml (300 ml) of chilled Buffer P3, mix immediately but gently, and incubate on ice for 5 min.
Precipitation is enhanced by using chilled Buffer P3 and incubating on ice.After addition of Buffer P3, the solution becomes cloudy and very viscous.To avoid localized potassium dodecyl sulfate precipitation, mix the solution gently, butthoroughly, immediately after addition of Buffer P3.Mix by inverting the tube 4-6 times.
4.Centrifuge at maximum speed in a microfuge for 10 min (or at 21,000 RPM in the F2402 rotor in the Beckman Avanti 30 centrifuge for 10 min).Remove supernatant promptly.Do step 5 during the centrifugation.
Before loading the centrifuge, the sample should be mixed again.Centrifugation should be performed at maximum speed in 1.5-ml or 2-ml microfuge tubes (e.g. 10,000-13,000 rpm in a microfuge).Maximum speed corresponds to 14,000-18,000 x g for most microfuges.After centrifugation, the supernatant should be clear.If the supernatant is not clear, a second, shorter centrifugation should be carried out to avoid applying any suspended or particulate material to the column.Suspended material (which causes the sample to appear turbid) will clog the column and reduce or eliminate flow.
- Remove a 50 µi sample and save it for an analytical gel (sample 1).
5.Put a QIAGEN-tip 20 in one of the collars and insert it into a 15-ml conical bottom tube.Equilibrate it by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow (takes 2-3 min).
Flow of buffer will begin automatically by reduction in surface tension due to the presence of detergent in the equilibration buffer.Allow the QIAGEN-tip to drain completely.The resin bed will retain some buffer and will not readily dry out.QIAGEN-tips can therefore be left unattended.Do not force out the remaining buffer.
6.Apply the supernatant from step 4 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow.
The supernatant should be loaded onto the QIAGEN-tip promptly.If it is left too long and becomes cloudy due to further precipitation of protein, it must be recentrifuged before loading to prevent clogging of the QIAGEN-tip.
-Remove a 5µl sample of the flowthrough and save for an analytical gel (sample 2).
7.Wash the QIAGEN-tip 20 with 4 x 1 ml Buffer QC.Each application of buffer takes about 2-3 min to go through.All of these can drain into the same 15-ml tube, and will be discarded.
Allow Buffer QC to move through the QIAGEN-tip by gravity flow.The first 2 ml are sufficient to remove all contaminants in the majority of plasmid DNA preparations.The second 2 ml ensure complete removal of contaminants in all situations, and will give consistent results in sequencing.The second 2 ml are particularly necessary when large culture volumes or bacterial strains containing large amounts of carbohydrate are used.It is particularly important not to force out residual wash buffers.Traces of wash buffer will not affect the elution step.
- Remove a 50 µl sample of the flowthrough and save for an analytical gel (sample 3)
