DNA extraction for RAPD's

This method has produced consistent results on my toughest template, the dreaded Pleopeltis polypodioides. It is a basic CTAB/phenol/chloroform type of DNA extraction protocol.

Prep:

  1. 10X CTAB
  2. 5M NaCl
  3. 0.25M EDTA
  4. 1M tris-HCl, pH 8.0

Mix Buffer:

  1. 2ml 10X (10%) CTAB (must be @ 65 deg. C)
  2. 80ul 0.25M EDTA
  3. 2.8ml 5M NaCl
  4. 1ml 1M tris-HCl, pH 8.0
  5. 20 ul mercaptoethanol
  6. 4.1ml deionized water

total = 10ml

  1. Grind about 20-50mg tissue in a sample vial (Fisher#02-544-5..sample cup) with 2.5mm glass beads and 200ul-400ul of buffer. Do not grind too vigorously. I use a motorized stirrer with an adjustablechuck, with a 5/8" glass rod inserted into the chuck. The action of the flat end of the glass rod against the beads grinds the sample. This method has been effective for tough to grind plant material.
  2. Remove the sample from the vial and place it in a 1.5ml microcentrifuge tube. Rinse the vial with 200ul-400ul sample buffer to remove as much material as possible to the microcentrifuge tube. The microcentrifuge tube should contain no more than 700ul of sample.
  3. Add Proteinase K, incubate for 1.5 hours at 65deg. C. extract with an equal volume of SEVAG (24 parts chloroform:1partisoamyl alcohol) mix by inversion.
  4. Incubate at 65 deg. C for 10 minutes.
  5. Incubate on ice for 10 minutes.
  6. Centrifuge 13,000 RPM for 5 minutes.
  7. Remove TOP layer to a new 1.5ml microcentrifuge tube.
  8. Extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1)
  9. Centrifuge 13,000RPM for 5 minutes
  10. Remove top layer to a new 1.5ml microcentrifuge tube
  11. Extract with an equal volume of SEVAG
  12. Centrifuge 13,000RPM for 5 minutes (until solution clear..but not necessarily colorless)
  13. Remove top layer to a new 0.5ml microcentrifuge tube
  14. Add 2/3 volume of ice cold isopropanol
  15. Chill tube to aid precipitation (overnight)
  16. Centrifuge 13,000RPM for 5 minutes
  17. Pour off isopropanol
  18. Wash with 76%EtOH, 10mM NH4OAc.
  19. Centrifuge 13,000RPM for 5 minutes.
  20. Pour off liquid, air dry pellet.
  21. Resuspend pellet in 250ul 10mM TE
  22. Determine DNA concentration, we use a spectrophotometer.
  23. Adjust volume to 250ng/ul, which is 10X recommended (Operon) concentration for RAPD's.
  24. Use 1:9 dilution to get 25ng/ul for PCR, add 1ul for reactions.

This method is a combination of several methods that I have tried, and seems to work better than anything else I have tried.