DNA Hybridization - Bacterial DNA
1. Seal uv-linked nylon filter in seal-a-meal heat-sealable bag with a 2 mm gap along 3 sides and a 3 cm open gap along 1 side.
2. Add 5 - 15 mL of HPB Hybridization Solution to bag.
3. Add 100 µL 100 mg/mL sonicated and denatured salmon sperm DNA to the solution.
4. Heat seal the fourth edge leaving a 2 cm gap. Incubate at 65° for 2 - 8 hrs with movement.
5. Decant prehybridization solution thoroughly.
6. Add 3 - 10 mL Hybridization Solution.
6. Add heat-denatured labeled probe and another 100 µL aliquot of Salmon Sperm DNA.
7. Heat seal the edge leaving a 2 mm gap.
8. Place this bag in another bag and seal the outer bag.
9. Incubate the double-bagged filter at 65° overnight with movement.
10. Cut open the corner of the hybridization bag and decant and discard the hybridization solution.
11. Cut open the hybridization bag along three edges and remove filter.
12. Add 10 mL Wash Solution:
1 mM Tris, pH 8.0 100 mL 1 M Tris, pH 8.0
1 % Sarcosyl 10 mL 10 % Sarcosyl
90 mL H2O
13. Swirl solution and immediately discard into radioactive waste..
14. Add remaining 90 mL Wash Solution. Wash at room temperature for 15 min with rotation.
15. Discard solution in radioactive waste.
16. Add 250 mL 1 mM Tris, pH 8.0. Rinse for 5 min at room temperature with rotation.
17. Discard rinse solution in radioactive waste.
18. Repeat rinse three addition times, these times discarding solutions down the sanitary sewer.
19. Wrap filter in Saran Wrap. Monitor with geiger counter.
20. Place wrapped filter on Whattman 1 MM paper in film cassette.
21. In darkroom, place appropriately sized X-Ray film, Kodak XAR-2 or equivilent, over filter and close cassette. Add intensifying screen on top of film if needed.
22. Expose approximately 1 hour for 1000 cpm to 48 hours for <100 cpm.
Last Update: Thursday, 19-Jun-2014 11:50:33 PDT
This page has been viewed 87 times.
eckofoid at ucdavis.edu