Plasmid Mini Purification Protocol（质粒纯化）

This protocol is for Mini (up to 20 µg) preparations of high-copy plasmid DNA from cultures of E. coli .

Important notes before starting

New users are strongly recommended to read Appendix A (page 21) at the end of this handbook before starting the procedure.

To ensure high yields of pure DNA , use no more than 3 ml LB culture for high-copy-number plasmids (e.g., pUC, pBluescript). For low-copy-number plasmids (e.g., pBR322), use no more than 10 ml LB culture and refer to the recommendations on page 13. We do not recommend the use of rich media such as TB or 2×YT for culture. When low-copy-number plasmids containing the ColE1 replication origin are prepared, the yield can be improved by amplification in the presence of chloramphenicol (34 mg/ml). They should then be treated as high-copy-number plasmids.

Add the provided RNase A solution to Buffer P1 before use (spin down RNase A briefly before use). Buffer P1 should then be stored at 2-8℃ and is stable for 6 months.

Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37℃.

Chill Buffer P3 at 4℃.

Optional: To confirm purification or to identify a problem, samples may be taken at specific steps for analysis on an agarose gel. Appropriate samples and volumes are indicated in the protocol below.

Procedure

1. Resuspend the bacterial pellet in 0.3 ml of Buffer P1. 在0.3 ml缓冲液P1中重悬细菌沉沉小球。

Ensure that RNase A has been added to Buffer P1. The bacteria should be resuspended completely, leaving no cell clumps.

2. Add 0.3 ml of Buffer P2, mix gently, and incubate at room temperature for 5 min. 加入0.3 ml缓冲液P2，轻轻混和，在室温下培养5 min。

After addition of Buffer P2, the solution should be mixed gently, but thoroughly, by inverting the tube 4-6 times. Do not vortex, as this will result in shearing of genomic DNA . The lysate should appear viscous. Do not allow the lysis reaction to proceed for more than 5 min. After use, the battle containing Buffer P2 should be closed immediately to avoid any reaction between the NaOH and CO2 in the air. If the buffer is left open for any length of time, it should be prepared fresh from stock solutions.

3. Add 0.3 ml of chilled Buffer P3, mix immediately but gently, and incubate on ice for 5 min. 加入0.3 ml冰冷的缓冲液P3，立即轻轻混和，在冰上培养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, but thoroughly, immediately after addition of Buffer P3. Mix by inverting the tube 4-6 times.

4. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove supernatant promptly. 用微量离心器在最大速度下离心10 min，迅速移出上清。

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.