Colony hybridization

1. Number each plate. Number the filters with a pencil and place three unique marks (e.g., one, two and three lines) around the edges of the filter.

2. Place filter onto plate and mark the corresponding lines onto the inside walls of the plate. Filter should become completely wet.

3. Lift the filter off the agar using forceps and lay the filter, colony side up, for about 2 min. on a doubled sheet of blotting paper soaked with denaturing solution in a dish (remove any excess solution).

4. Put the blotting paper and filters on top of two more dry sheets of blotting paper and microwave at full power for 3 min, 30 sec.

5. Wet filters in 2X SSC. Prehybridize in Church buffer for 30-60 min. Add labeled probe and hybridize 6+ hrs.

6. Wash filters with 2X SSC, 0.1% SDS at RT for 10 min, then at 65°C for 15-30 min.

7. Stain the colonies on the filters to aid identification of positive by dissolving a spatula-tip full of Coomassie blue in 1 mL of 95% ethanol.

8. Add this to 10 ml of 0.1X SSC, 0.01% SDS in a petri dish.

9. Incubate the filters in the stain at RT for about 20 min.

10. Remove the stain solution, which can be retained and reused.

11. Wash the filters with 0.1X SSC, 0.01% SDS to remove background stain.

12. Blot off excess liquid and expose the filters to X-Ray film.

Denaturing Solution (50 mM NaOH, 0.5% SDS), 50 ml

0.25 ml 10 N NaOH
1 ml 25% SDS
48.75 ml ddH2O
Staining solution, 20 mL

0.1 mL 20X SSC, pH 7
0.08 mL 25% SDS
19.82 mL ddH2O