Plasmid

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.

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。）

Precipitation of DNA with isopropanol should be carried out with all solutions equilibrated to room temperature in order to minimize salt precipitation. Isopropanol pellets have a glassy appearance and may be more difficult to see than the fluffy, salt-containing pellets that result from ethanol precipitation. Marking the outside of the tube before centrifugation allows the pellet to be easily located.

10. Wash DNA with 1 ml of 70% ethanol, air-dry for 5 min, and redissolve in a suitable volume of buffer. 用1 ml 70%酒精洗涤DNA，在空气中干燥5 min，用适当体积缓冲液重新溶解。

（可用移液器从沉淀小球对面移去酒精，后在通风处或超净工作台送风条件下风干，但不宜过干，过干很很难溶解）

The DNA pellet should be washed briefly in 70% ethanol, and then recentrifuged. The 70% ethanol serves to remove precipitated salt, as well as to ｒｅｐｌａｃｅ isopeopanol with the more volatile ethanol, making the DNA easiser to redissolve. A second wash with room-temperature 70% ethanol may improve results in more sensitive applications, such as transfection. After careful and complete removal of ethanol, the pellet should be air-diried briefly (approximately 5 min) before redissolving in an appropriate volume of TE buffer. Overdrying the pellet will make the DNA difficult to redissolve. Resuspend the DNA pellet by rinsing the walls to recover all the DNA. Pipetting the DNA up and down to promote resuspension may cause shearing, and should be avoided. DNA dissolves best under slightly alkaline conditions; it does not easily dissolve in acidic buffers.

Determination of yield

To determinate the yield, DNA concentration should be determined by both UV spectro-photometry and quantitative analysis on an agarose gel.

Analytical gel (optional)

To analyze the purification procedure as shown in Figure 2 (Appendix A, page 27), precipitate samples 1-4 (from steps 4-8) with 35 µl of isopropanol. Rinse the pellets with 70% ethanol, drain well, air-dry, and resuspend in 10 µl of TE, pH 8.0. Use 2 µl of each for analysis on a 1% agarose gel.

Purification of cosmids and low-copy-number plasmids

From: QIAGEN Plasmid Mini Handbook

Cosmids and low-copy-number plasmids often require large culture volumes to yield significant amounts of DNA. A few modifications to the protocol will avoid overloading the system. The following procedure is recommended for optimal results.

Note: Many cosmids yield less than 1 µg DNA per ml of culture. Quantify the amount of DNA obtained in a pilot experiment by comparison to standards on an agarose gel. Adjust LB culture volumes accordingly (maximum 10 ml). If culture volumes greater than 10 ml are required, use the appropriate QIAGEN Plasmid Midi, Maxi, Mega, or Giga Kit.

1. Resuspend the bacterial cells in an appropriate volume of Buffer P1. Use 9.3 ml of P1 for every 3 ml of LB culture.

2. Increase the volume of Buffers P2 and P3 to match the volume of P1 used in the previous step.

3. Centrifuge at maximum speed (10000-13000 rpm) for 10 min at room temperature. Transfer the supernatant promptly to a fresh tube and recentrifuge for 10 min at maximum speed. Transfer the supernatant to a fresh tube. After centrifugation the supernatant should be clear. Suspended or particulate material (causing the sample to be turbid) will clog the column and reduce or eliminate flow. Alternatively, the centrifugation process may be replaced by use of a QIAfilterTM Midi Cartridge.

4. Precipitate the DNA in the supernatant with 0.7 volumes room-temperture isopropanol. Centrifuge at maximum speed for 30 min at room temperature ande carefully decant the supernatant.

5. Redissolve the DNA pellet in a small volume of TE, pH 7.0, and add Buffer QBT to obtain a final volume of at least 1 ml. Apply the sample to a previously equilibrated QIAGEN-tip 20.

Note: To compare the amount of plasmid in the culture with amount obtained after purification, remove a comparable aliquot (11%) of the final purified sample and quantify the amount of plasmid each contains by comparison to standards on an agarose gel. A recovery of 80-% is typical.

6. Continue with the standard purification protocol at step 7 (page 11).

Note: An extra 5-10% increase in recovery of cosmid DNA may be obtained by heating Buffer QF to 65℃ before elution (step 8).

Purification of BACs, PACs, PIs, and other large plasmids

BACs, PACs, PIs, and other large plasmid constructs can be purified on QIAGEN-tip 20 with a few modifications to the standard protocol. Recommendations are give below. Cultures should be grown overnight in 5 ml LB medium.

1. Resuspend bacterial cells in 0.6 ml Buffer P1, and also increase volumes of lysis buffers P2 and P3 to 0.6 ml.

2. Elute DNA using 2×0.4 ml pre-warmed (65℃) Buffer QF.

3. After isopropanol precipitation, redissolve DNA in 20 µl 10 mM Tris-Cl, pH 8.5, incubating overnight at 4℃ to redissolve completely.

（此步可用TE缓冲液（pH 8）溶解DNA替代）。