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S U P P O R T
 

Protocol for Production of Single-stranded Circular DNA Molecules from Supercoiled Double-stranded Plasmids in vitro (with Nb.Bpu10I)

  1. Add the following components to a  reaction tube:
    Plasmid containing Bpu10I recognition sequence (20 µg) 20- 356 µl
    10X Buffer R 40 µl
    Nb.Bpu10I 4 µl (20 u)
    Water, nuclease-free to 400 µl
  2. Vortex the tube and spin in a microcentrifuge for 3-5 seconds.
  3. Incubate at 37ºC for 1 hour .
  4. Add ½ volume of phenol (200 µl) and ½ volume of chloroform/isoamyl alcohol (24:1) (200 µl), vortex for 10 sec. and centrifuge at 10,000 rpm for 5 minutes.
  5. Transfer the upper aqueous phase to a fresh tube and add 1 volume (400 µl) of chloroform/isoamyl alcohol (24:1). Vortex and centrifuge for 5 minutes.
  6. Repeat step 5 twice more.
  7. Transfer the upper aqueous phase to a fresh tube. Add 1/10 volume of 3M sodium acetate and 2.5 volumes of ice-cold ethanol. Mix and incubate at -20ºC for 1 hour.
  8. Centrifuge at 10,000 rpm for 10 minutes.
  9. Pour off the supernatant and carefully wash the pellet with 200 µl of 75% ice-cold ethanol. Dry the pellet.
  10. Dissolve DNA in 50 µl of water, nuclease-free.
  11. Treat with Exonuclease III  by adding the following components:
    1X reaction buffer for ExoIII 25 µl
    Exonuclease III 6 µl (1200 u)
    Water, nuclease-free to 200 µl
  12. Mix and incubate at 30ºC for 10 min. Stop the reaction by heating at 70ºC for 10 min .
  13. Extract the reaction mixture with phenol/chloroform as described in stages 4-6, precipitate DNA as described in steps 7-9 and dissolve in 10-30 µl of deionized water. This solution should contain single-stranded DNA, suitable for DNA sequencing, site-specific mutagenesis, differential display, etc.
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Updated gegužės 09, 2007 16:39