3.3.2.2. Restriction enzyme digestion

  1. Aseptically remove a plug from the TE80 buffer and place onto the inner surface of a sterile petri dish.
  2. Using a plastic ruler under the petri dish, cut two 2 mm slices from the plug with a sterile razor blade.
  3. Place the two plug slices into 1 ml TE80 buffer in a 1.5 ml microcentrifuge tube.
  4. Wash the slices 2X with 1 ml TE80 buffer (30 min for each wash with gentle agitation).
  5. Quickly rinse the slices with 500 µl restriction digestion buffer (without restriction enzyme).
  6. Add 100 µl of XbaI Digestion Mixture  (see recipe below) and incubate 3 h at 37oC with gentle agitation.
  7. Remove digestion mixture solution.
  8. Rinse slices 2X with 1 ml 0.5X TBE Buffer (see recipe below).
  9. Suspend the plug slices in 1 ml 0.5X TBE Buffer.
  10. Store overnight 4oC.

 

XbaI Digestion Mixture (10 samples):

  • 10X New England BioLabs Buffer 4 (100 µl)
  • 100X BSA (10 µl)
  • XbaI enzyme (20 µl) New England BioLabs (20,000 U/ml)
  • H2O (870 µl)

 

5X TBE Buffer (500 ml):

  • Tris Base (54 g)
  • boric Acid (27.5 g)
  • 0.5 M EDTA (pH 8.0) (20 ml)
  • H2O to 500 ml volume