Plasmid Mini Purification Protocol

Important Notes Before Starting

• New users are strongly recommended to read the QIAGEN handbook before starting the procedure.

• Before using the kit for the first time, dissolve the lyophilized RNAse A provided Buffer PI . Buffer PI should then be stored at 4°C 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°C.

• Pre-chill Buffer P3 at 4°C. It should be in the refrigerator, along with P1.

Day before doing plasmid prep:

• Put 4 ml of LB (with appropriate antibiotic added) into a 50-ml conical bottom tube. Add one colony of the desired bacterial/plasmid culture and incubate at 37° with shaking overnight. It’s essential to use selective media, as otherwise a lot of your bacteria will lose the plasmid.

Before starting prep:

a. Get a bucket of ice and put P1 and P3 in it.

b. Take out one milliliter of each liquid culture and put it in a cryovial with one milliliter of glycerol freezedown solution (see recipe). Mix thoroughly, label with plasmid name, date, and initials, and put in -85° freezer.

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

1. Resuspend the bacterial pellet in 0.3 ml (300 ml) of Buffer P1. Vortex to mix thoroughly, then pipette the suspension into a 1.5 ml microcentrifuge tube.

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

2. Add 0.3 ml (300 ml) of Buffer P2 , Mix gently, and incubate at room temperature for 5 min.

After addition of Buffer P2, the solution should be mixed gently, but thoroughly, by inverting the tube A-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 pro for more than 5 min. After use, the bottle containing Buffer P2 should be closed immediately to avoid any reaction between the NaOH and C0₂ 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 (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, but thoroughly, 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)

8. Move the QIAGEN column to a sawed-off 15-ml tube (in the QIAGEN materials box) and press the tube down over a new, labelled 1.5 ml microfuge tube so that the lid folds down over the edge of a microfuge tube rack. Elute DNA into the microfuge tube with 0.8 ml (800 ml) Buffer QF.

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 (560 ml, if you used 800 ml elution volume) of room-temperature isopropanol. Centrifuge immediately @ 21,000 rpm for 15 min in the Beckman Avanti 30 (F2402 rotor), and pour off the supernatant.

Precipitation of DNA with isopropanol should be carried out with all solution 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 flu salt-containing pellets that result from ethanol precipitation. It is a good idea to mark the outside of the tube before centrifugation, so that the pellet can be more easily located.

10. Add 1 ml of cold 70% ethanol to the tube with the pellet, being careful not to disturb the pellet. Centrifuge immediately @ 21,000 rpm for 10 min in the Beckman Avanti 30 (F2402 rotor). Dry for 2-3 min in the speed vac on low setting. Check after 2 min for smell of alcohol; if none, add 20 ml of sterile, distilled, deionized water. Otherwise dry a minute longer and check again. The pellet should not be completely dry -- a small amount of water will remain after all alcohol is gone. If you Speed-vac it to dryness, it will be very hard to redissolve.

The DNA pellet should be washed briefly in 70% ethanol, and then recentrifuge The 70% ethanol serves to remove precipitated salt, as well as to replace isopropanol with the more volatile ethanol, making the DNA easier to redissolve. A second was with room-temperature 70% ethanol may improve results in more sensitive application such as transfection and sequencing. After careful and complete removal of ethanol the pellet should be air-dried before redissolving in a appropriate volume of TE buffer (use high-quality water instead if it’s to be sequenced). 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. The DNA may also be difficult to dissolve if it is too acidic. DNA dissolves best under slightly alkaline conditions.

11. To determine the yield, measure DNA concentration in a UV spectrophotometer. (followed by analysis on an agarose gel). Readings on a spectrophotometer are not always accurate, particularly if a single wavelength measurement is taken rather than a scan.

Analytical gel (optional): to analyze the purification procedure as shown in Figure 2(Appendix A, page 19), precipitate samples 1-4 (from steps 4-8) with 35 µ isopropanol. Rinse the pellets with 70% ethanol, drain well, air-dry, and resuspend 10 µl of TE, pH 8.0. Use 2 µi of each for analysis on a 1 % agarose gel (1).