[Insert Signature here. You must be signed in to do this]

This protocol is designed for purification of up to 20 µg high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli grown in LB medium, using QIAprep 2.0 spin columns on QIAvac 24 Plus or other vacuum manifolds with luer connectors.

Here you cleanly list all of the reagents and equipment in our lab or elsewhere involved in performing this protocol, with links to our wiki reagent or equipment pages if necessary (make as needed the first time you enter a protocol- a protocol is not complete without completed links to all reagents and equipment). Please include this information in a table

Plasmid DNA Purification using the QIAprep Spin Miniprep Kit and a Vacuum Manifold

Note: All protocol steps should be carried out at room temperature (15–25°C). Procedure 1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube gently 4–6 times. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, immediately after addition of Buffer N3 mix the solution gently but thoroughly. Large culture volumes (e.g. ≥ 5 ml) may require inverting up to 10 times. The solution should become cloudy. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.

During centrifugation, prepare the vacuum manifold and QIAprep 2.0 spin columns: QIAvac 24 Plus (see pages 14, and 16-18):

■ Ensure that the vacuum source is connected to the upper threaded hole of the QIAvac 24 Plus and the lower threaded hole is tightly sealed using the screw cap.

■ If using the QIAvac Connecting System, connect the system to the manifold and vacuum soured as described in the QIAvac 24 Plus Handbook.

■ Insert up to 24 spin columns into the luer slots of the QIAvac 24 Plus. Close unused luer slots with luer plugs.

Other vacuum manifolds: Follow the supplier’s instructions. Insert each QIAprep 2.0 column into a luer connector

5. Apply 800 µl of the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.

6. Switch on vacuum source to draw the solution through the QIAprep 2.0 spin columns, and then switch off vacuum source.

7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Switch on vacuum source. After the solution has moved through the column, switch off vacuum source. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step.

8. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Switch on vacuum source to draw the wash solution through the column, and then switch off vacuum source.

9. Transfer the QIAprep 2.0 spin columns to a microcentrifuge tube. Centrifuge for 1 min. Important: This extra spin is necessary to remove residual Buffer PE. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Vacuum notes

■ Switch off vacuum between steps to ensure that a consistent, even vacuum is applied during manipulations. ■ Wear safety glasses when working near a manifold under pressure. ■ For safety reasons, do not use 96-well plates that have been damaged in any way. ■ For the QIAprep 96 Turbo miniprep procedure, the negative pressure (vacuum) should be regulated before beginning the procedure by applying the vacuum to the appropriate number of empty QIAprep modules (indicated in Table 3) on the QIAvac manifold. The vacuum pressure is the pressure differential between the inside of the manifold and the atmosphere (standard atmospheric pressure: 1013 millibar or 760 mm Hg) and can be measured using a vacuum regulator (see ordering information, page 40). Vacuum recommendations are given in negative units (Table 3) to indicate the required reduction in pressure with respect to the atmosphere. Table 4 provides pressure conversions to other units. ■ Use of a vacuum pressure lower than recommended may reduce DNA yield and purity

Guidelines for QIAvac manifolds

QIAvac 24 Plus and QIAvac 96 facilitate DNA minipreps by providing a convenient modular vacuum manifold for use with the QIAprep system. The following recommendations should be followed when handling QIAvac manifolds. ■ QIAvac manifolds operate with a house vacuum or Vacuum Pump (e.g., Vacuum Pump, cat. no. 84010 [USA and Canada], 84000 [Japan], or 84020 [rest of world]). ■ Always store QIAvac manifolds clean and dry. To clean, simply rinse all components with water and dry with paper towels. Do not air dry, as the screws may rust and need to be replaced. Do not use abrasives or solvents. ■ Always place the QIAvac manifold on a secure bench top or work area. If dropped, the manifold may crack. ■ The components of QIAvac manifolds are not resistant to ethanol, methanol, or other organic solvents (Table 5). Do not bring solvents into contact with the vacuum manifold. If solvents are spilled on the unit, rinse thoroughly with distilled water. Ensure that no residual Buffer PE remains in the vacuum manifold. QIAprep Miniprep Handbook 06/2015 17 ■ To ensure consistent performance, do not apply silicone or vacuum grease to any part of a QIAvac manifold. The spring lock on the top plate and the self-sealing gasket (QIAvac 96) provide an airtight seal when vacuum is applied to the assembled unit. To maximize gasket lifetime, rinse the gasket free of salts and buffers after each use and dry with paper towels before storage.