RPM^® SPIN MIDI Protocol
Rapid Pure Midiprep Kit
Revision No. 2005-999-6J04W

The RPM SPIN MIDI Kit is designed for rapid isolation and purification of double stranded plasmid DNA from bacterial cells. It delivers high yields of supercoiled plasmid DNA with minimum effort and time. The resulting DNA is suitable for transfection/transformation and enzymatic manipulations including sequencing, restriction enzyme digestion, in vitro transcription*, and PCR.

1. Pellet culture in a tabletop centrifuge at 4000 rpm for 5 minutes and decant media. Resuspend cells in 500 µl H₂O by vortexing and transfer to a 2 ml microcentrifuge tube. Spin 30 seconds and decant supernatant. (Cell pellet can be stored at -20°C at this point until ready to process. Thaw 5-10 minutes at 37°C and continue with step 2).


2. Add 200 µl Pre-Lysis Solution and mix by vortexing until cells are completely resuspended. (This is essential for efficient cell lysis and maximum DNA yield).


3. Add 400 µl Alkaline Lysis Solution (Before use, dissolve precipitated SDS by warming, if necessary) and gently invert 15 times. Optional: incubate < 5 minutes at room temperature.


4. Add 300 µl ice cold Neutralizing Solution and shake vigorously 3-5 times until a uniform white precipitate forms. Optional: incubate 5 minutes on ice. Spin 5 minutes and transfer supernatant to a new 2 ml microfuge tube (Avoid transferring precipitate).



"GENECLEAN" Purification of Plasmid DNA

High Copy Vectors: (pUC, pTZ, pGEM, pBS, etc)

5. Add GLASSMILK SPIN Buffer* (1.2 ml for high and 0.9 ml for low copy vectors); invert gently several times to ensure a uniform suspension and incubate 5 minutes at room temperature with continuous mixing for efficient DNA binding to GLASSMILK. *Mix GLASSMILK Spin Buffer completely before each use.


6. Spin the GLASSMILK/DNA complex 2-5 seconds and decant supernatant. (Plasmid DNA is bound to the GLASSMILK. The supernate contains proteins, metabolites, and degraded RNA). Add 500 µl Wash Solution (add ethanol before first use); resuspend the GLASSMILK/DNA complex by gently stirring with a pipet tip, followed by pipetting up and down (complete resuspension is not essential for efficient washing). Transfer GLASSMILK/DNA solution to a kit-supplied SPIN Filter.


7. Spin 20 seconds to lower the level of wash solution to the level of the pellet without drying it; empty Catch Tube and add 500 µl Wash Solution and spin again. Spin 2-5 minutes to "dry" the filter contents. Transfer the filter to a new kit-supplied Catch Tube. (Do not let the filter dry completely as the membrane might crack during centrifugation).



DNA Elution

High Copy Vectors:
Elute with 500 µl Elution Solution, H₂O, or TE

Low Copy Vectors:
Elute with 100-200 µl Elution Solution, H₂O, or TE (Elution Solution is RNase-, DNase-, Pyrogen-free water)




8. Add appropriate volume of Elution Solution, autoclaved H₂O, or TE and resuspend the GLASSMILK/DNA complex by gently stirring with a 200 µl pipet tip or by gentle finger tapping. (Do not vortex. Do not push on the membrane with pipet tip to prevent rupture. Complete resuspension of the GLASSMILK/DNA complex ensures maximal DNA elution). Spin 2-5 minutes to collect the DNA in the Catch Tube and discard the Filter containing used matrix. Store DNA at 4-20°C. DNA is ready for use.