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 RNA se A provided Buffer PI . Buffer PI should then be stored at 4℃ 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℃.

•Pre-chill Buffer P3 at 4℃.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 m l) of Buffer P1.Vortex to mix thoroughly, then pipette the suspension into a 1.5 ml microcentrifuge tube.

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

2. Add 0.3 ml (300 m l) 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 m l) 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, butthoroughly, 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).