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.
