activity using X-gal
by Michael Koelle, adapted from Laird Bloom, Jeff Way, & Michael Basson
4/6/94
1. Grow up a small plate of worms. Some protocols call for using ß-gal[-] bacteria on the plates (e.g. DH5 alpha) which may eliminate some staining due to bacteria in the pharynx or gut.
2. Wash the worms off the plate with ~2 ml water, transfer with a pasteur pipette to a 15 ml centrifuge tube, add water to ~5 ml, spin in a clinical centrifuge ~1 min., and remove all but ~400 ul supernatant with a pasteur pipette. Transfer the worms with a pasteur pipette to a 500 ul eppendorf tube, spin 3K for 1 min. in a variable speed microfuge, and remove as much of the supernatant as possible using a pipetteman.
3. Cap the tubes and freeze in liquid nitrogen. Open the caps and stick in a vacuum jar or speed vac for about 45 minutes to lyophilize.
4. Add ~250 ul cold acetone, and let sit in the freezer 3 min. Remove as much acetone as possible with a pipetteman, and speed vac off the rest.
5. ß-gal staining solution:
For a final volume of 1 mL, add in order:
620 ul ddH20
250 ul 0.8 M Na-phosphate buffer pH7.5
1 ul 1 M MgCl2
4 ul 1% SDS
100 ul 100 mM Redox buffer (see below, keep stocks at -20deg. C)
15 ul 5 mg/mL kanamycin (keep stock at -20deg. C)
2 ul 1 mg/ml DAPI (keep stock wrapped in foil at -20deg. C)
8 ul 5% X-Gal in dimethyl formamide. (keep at -20deg. C)
Add the X-Gal last, and then vortex quickly to avoid precipitation of the X-Gal.
Staining solution can be kept for at least 2 days (wrapped in foil at 4deg.).
Redox buffer is made fresh each time by mixing equal volumes of the following stock solutions:
100 mM Potassium Ferricyanide
100 mM Potassium Ferrocyanide
Keep both stocks at -20deg.. CAUTION: Wear gloves, these solutions are toxic (contain cyanide).
6. Add ~200 ul stain solution to the worms. Can stain at room temp or 37deg.. Periodically take out a small amount of the worm suspension, drop on a microscope slide, and examine under a high power dissecting scope to monitor progress of the staining. Some constructs require 24 hrs for the blue color to develop.
7. When the stain is satisfactory, wash the worms a couple times in PBS to remove the staining solution.
8. To view the worms, mount on an agar pad and examine on the Normarski scope.
