RPM^® AFS Protocol
Rapid Pure Midiprep for "LongRead" Automated Fluorescence Sequencing
Revision No. 2072-999-5L02W

The RPM AFS Kit is designed for high throughput template preparation for automated fluorescence sequencing. It delivers high yields of supercoiled plasmid DNA with minimum effort and time. The resulting DNA is also suitable for transfection/transformation and enzymatic manipulations including sequencing, restriction enzyme digestion, in vitro transcription with RNase inhibitor, and PCR.

1. Modified Alkaline lysis; RNA Degradation; Precipitation of Proteins, Cell Debris and Genomic DNA.
   1. Pellet culture in a tabletop centrifuge at 4000 rpm for 5 minutes and decant media. Resuspend cells in 1 ml H₂O by vortexing; transfer to a 2 ml microfuge tube; spin for 30 seconds and decant supernate (cell pellet can be stored at -20°C until ready to process. Thaw 5-10 minutes at 37°C and continue with step b).
   2. Add 200 µl Pre-Lysis Buffer; mix by vortexing until the cells are completely resuspended. (Complete resuspension is essential for efficient cell lysis and maximal 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. Spin 5 minutes at room temperature and transfer supernate to a new 2 ml tube.
2. "GENECLEAN" Purification of Plasmid DNA
   1. Add 500 µl AFS GLASSMILK; invert gently several times to ensure a uniform mixture, and incubate 5 minutes at room temperature with continuous mixing for efficient DNA binding to GLASSMILK.
   2. Spin 2 seconds and decant supernate. (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 pipette 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.
   3. 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 2-5 minutes to "dry" filter contents. Transfer the SPIN Filter to a new kit supplied Catch Tube (do not let the filter completely dry as the membrane might crack during centrifugation).
   4. Add 100-200 µl Elution Solution (RNase/DNase/pyrogen-free H₂O), and mix the GLASSMILK/DNA complex into a slurry by gently stirring with a 200 µl pipet tip or by gentle finger tapping (Do not vortex. Even though the Spin Filter membrane is sturdy and will not easily rupture, do not push on the membrane with pipet tip. 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 SPIN Filter containing used matrix. Store DNA at 4-20°C. DNA is ready for use.

Adjust DNA template concentration, if necessary, by ethanol precipitation.