RPM^® Protocol
Rapid Pure Miniprep Kit
Revision No. 2070-999-6H02W

The RPM^® Kit delivers high yields of supercoiled plasmid DNA with minimum effort and time. Multiple samples can be processed rapidly by using 1.5 ml of bacterial culture. With practice, the procedure can be completed in less than 12 minutes, yielding pure plasmid DNA suitable for enzymatic manipulations including DNA sequencing, restriction enzyme digestion, in vitro transcription, and PCR. Reagents and materials supplied by the user are 100% ethanol, sterile water or TE buffer, 1.5ml microcentrifuge vials (one for each miniprep), and a microcentrifuge. The reagents and materials provided with the kit are for research use only.

1. Spin 1.5 ml of bacterial culture in a microcentrifuge vial (user-supplied) for 30 seconds; remove and discard supernatant. If use of more than 1.5 ml culture for this miniprep is desired, add more culture; re-spin; remove and discard supernatant. (Pour off supernatant and then remove the remaining few microliters with a pipet tip).
2. To resuspend the cell pellet, add 50 µl Pre-Lysis Buffer and mix by vortexing by pipetting up and down until completely suspended.
3. Add 100 µl Alkaline Lysis Solution directly into the cell suspension and mix by inverting the tube several times. Before proceeding to step 4, wait 1 minute to allow lysis to complete (solution should be clear).




This is a critical step in the procedure and affects yield and purity of the plasmid DNA; the cells must be homogeneously lysed and some host cells lyse easier than others. For optimum consistency of lysis from one cell type to another, be certain the Alkaline Lysis Solution is 22-25°C and that the SDS is in solution.




4. Add 100 µl Neutralizing Solution and mix by briefly vortexing. A white precipitate will form consisting of cell membranes, proteins, and chromosomal DNA. Spin in a microcentrifuge for 2 minutes to pellet white precipitate.
   1. Prepare a SPIN^TM Filter for each sample by adding 250 µl (mix suspension completely before each use) GLASSMILK^® Spinbuffer to the SPIN^TM Filter chamber and placing it in a Catch Tube.
   2. Remove supernatant from sample while avoiding white pellet, and transfer to a prepared SPIN^TM Filter (see step 4a).
5. Pipet up and down a couple of times to mix and, after 1 minute, spin for 1 minute to collect liquid in the bottom of the Catch Tube.
6. Add 350 µl Wash Solution (remember to add an equal volume of ethanol before first use)* to SPIN^TM Filter and spin for 1 minute. Pour off liquid in collection vial and spin again for 1 minute to drive last of the liquid out of the SPIN^TM Filter. Transfer SPIN^TM Filter to a new Catch Tube. Discard other tube.
7. To elute plasmid DNA from the SPIN^TM Filter, add 50 µl of autoclaved sterile water or TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA); vortex briefly (at no more than half-speed) to resuspend GLASSMILK; and spin for 30 seconds to collect DNA in the bottom of the Catch Tube. Remove Catch Tube from microcentrifuge; remove and discard SPIN^TM Filter. Close Catch Tube containing plasmid DNA and store at +4°C or -20°C. Note: The yield of plasmid DNA is dependent on the bacterial host and the plasmid copy number. The plasmid DNA is now ready for further manipulation.