Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes (e.g., 10000-13000 rpm in a microcentrifuge). Maximum speed corresponds to 14000-18000×g for most microcentrifuges. After centrifugation, the supernatant 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 µl sample from the cleared lysate and save it for an analytical gel (sample 1).
5. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow. 利用1 ml缓冲液QBT对QIAGEN-tip 20尖管进行平衡处理，让其在重力作用下从柱中流出。
Place QIAGEN-tips into a QIArack over the waste tray or use the tip holders provided with each kit. 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. 将第4步中的上清倒入QIAGEN-tip 20尖管，让其在重力作用下进入树脂。
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, is must be recentrifuged before loading to prevent clogging of the QIAGEN-tip.
Remove a 50 µl sample of the flow-through and save for an analytical gel sample 2.
7. Wash the QIAGEN-tip 20 with 4×1 ml Buffer QC. 用4×1 ml缓冲液QC对QIAGEN-tip 20尖管进行清洗。
Allow Buffer QC to move through the QIAGEN-tip by gravity flow. The first 2 ml is sufficient to remove all contaminants in the majority of plasmid DNA preparations. The second 2 ml ensures complete removal of contaminants, and will also ensure consistent results in sequencing. (The second 2 ml is particularly necessary when large culture volumes or bacterial strains producing 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 combined wash fractions and save for an analytical gel (sample 3).
8. Elute DNA with 0.8 ml Buffer QF. 用0.8 ml缓冲液QF稀释DNA。
Place the upper part of a QIArack over the lower rack fitted with clean 1.5 ml or 2 ml microcentrifuge tubes and collect the samples in the tubes. Alternatively, use the tip holders provided. Flow begins when Buffer QF is added. Drain the QIAGEN-tip by allowing it to empty by gravity flow.
Remove a 50 µl sample of the eluate and save for an analytical gel (sample 4).
9. Precipitate DNA with 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of room-temperature isopeopanol. Centrifuge immediately at ≥10000 rpm for 30 min in a microcentrifuge, and carefully decant the supernatant. 用0.7体积（每0.8 ml稀释体积用0.56 ml）的isopeopanol在室温下沉淀DNA。 立即用微量离心机中在≥10000 rpm 转速下离心30 min。
（此步以后在离心机上操作要注意离心管的放置方向，保持沉淀方向一致或作好标记。以利于用移液管移吸上清，不会搅动沉淀。另外，抽吸剩下少量溶液可再离心一次。）
