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