RPM^® 1G
Rapid Pure Maxiprep Kit
Revision No. 2077-999-6D03W

The RPM 1G 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. Modified Alkaline lysis; RNA degradation; Precipitation of proteins, cell debris and genomic DNA:
   1. Pellet culture at 6000 xg for 5-10 min (e.g. 6000 rpm in GSA rotor) and discard media. (Optional: Store pellet at -20°C until ready to process. Thaw 5-10 min at 37°C and continue with step b).
   2. Resuspend cells in 10 ml H₂O by vigorous shaking and transfer to a 50 ml centrifuge tube. Add 1 ml Cell Wash Concentrate; swirl to mix and incubate 5 min at room temperature. Spin 5 min at 6000 x g and decant supernate. The wash dramatically reduces pyrogen levels in purified plasmid DNA.
   3. Add 7 ml Pre-Lysis Buffer and mix by vigorous shaking until the cells are completely resuspended (this is essential for efficient cell lysis and maximal DNA yield).
   4. Add 7 ml Alkaline Lysis Solution 1G (Before use, dissolve precipitated SDS by warming, if necessary) and gently invert 15 times. Optional: incubate < 5 min at room temperature.
   5. Add 7 ml ice cold Neutralizing Solution and shake vigorously 3-5 times until a uniform white precipitate forms. Incubate 10-20 min on ice; spin 10 min at 15,000 xg at 4°C (e.g., 12,000 rpm in SS-34 rotor) and transfer supernate to a clean 50 ml tube by decanting through a funnel lined with a kit-supplied Sieve Material* to remove floating debris.

   *Sieve Material is reusable. Rinse well under running tap and blot dry after each use.

   6. Add 100 µl RNase Mixx; invert 4 times to mix and incubate 10-15 minutes at 55°C.



2. "GENECLEAN" purification of plasmid DNA
   
   1. Add 20 ml GLASSMILK 1G; invert gently several times to ensure a uniform mixture and incubate 5 min (room temperature) with occasional mixing for efficient DNA binding to GLASSMILK. (Mix GLASSMILK 1G completely before each use. If crystals form, dissolve at 60°C). Spin at 600 x g for 1-2 min (2000 rpm in both an SS34 rotor and tabletop centrifuge) and discard the supernate. (Plasmid DNA is bound to GLASSMILK. Supernate contains proteins, metabolites, and degraded RNA).
   2. Add 10 ml of user-supplied 1:1 acetone/ethanol solution and resuspend the GLASSMILK/DNA complex by first dislodging the pellet with a pipet and then shaking back and forth. Add 10 ml of Wash Solution (remember to add ethanol before first use) and invert to mix. Spin 2 minutes in a tabletop centrifuge at 3-5,000 rpm and decant the supernate (it is not necessary to dry the GLASSMILK/DNA complex for efficient elution in H₂O, step 2c).
   3. Add 10 ml H₂O and resuspend the pellet as above (step 2b). Spin in a tabletop centrifuge at 3-5,000 rpm for 5 minutes.
   4. Transfer supernate to a 15 ml centrifuge tube and spin at 5000 rpm for 5 minutes to pellet silica particles and transfer supernatant to a 50 ml centrifuge tube.
   5. Add 400 µl 5 M NaCl, 10 ml isopropanol; and invert a couple of times to mix. Spin 10-20 minutes and decant the supernate. Spins should be at 15000 x g at 4°C (e.g. 12,000 rpm in SS-34 fixed angle rotor or 10,000 rpm in an HG-4 swinging bucket rotor. The use of a swinging bucket rotor will ensure that the DNA pellet is collected at the bottom of the tube and not partially on the wall of the tube; also, see note below). Wash the DNA pellet with 5-10 ml 80% ethanol; spin 5 minutes and carefully decant the supernate (careful; pellets can dislodge from tube at this point). Air dry DNA pellet for 5 minutes at room temperature and dissolve in 500 µl Elution Solution (nuclease/pyrogen-free H₂O) or TE. Store DNA at 4-20°C. DNA is ready for use.
   
   
   

   Note: Be certain to recover DNA that is smeared on the tube wall during step e above, especially if using a fixed angle rotor. Over-drying the pellet will make the DNA difficult to resuspend. A 5 minute incubation at 55°C will accelerate dissolving of the DNA pellet. Pipetting up and down to dissolve the precipitated DNA may cause shearing; however, gentle pipetting using a 1 ml pipette tip with 1-3 mm of the tip cut off is okay.

   
   
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