Southern Hybridization
Theory:
Materials & reagents:
Protocol:
DAY 1: Construct DNA probe as directed for BRL RadPrime DNA labeling system. Boil water in microwave to denature for 5 min., thaw 32P for 30 min. Perform the following steps in late afternoon for overnight incubation. 1. Remove Southern transfer set up. 2. Place membrane on 3 MM whatman paper and air dry until no moisture is present. 3. For Hybond (Amersham) membrane, crosslink DNA by exposure to UV radiation for 5 min. For MSI membrane, bake a 80!C for 2 hr. 4. Preheat hybridization oven to 65!C. Prewet the hybridization membrane in a tray containing SSC. 5. Roll membrane into a tight roll, clockwise (@ roll away from you). 6. Transfer the roll into oven bottle: position bottle to left of roll, insert rolled membrane, add 30 ml 2X SSC, and cover bottle. @==0 --> :::::::::] ----> @::::::::0:] 7. Unroll membrane by placing bottle on flat surface, gently rocking, and rolling counterclockwise (towards you).No air bubbles should be visible! Bubbles can be removed by pushing with long tweezers; small bubbles are okay. Otherwise, remove roll and repeat procedure. 8. Remove SSC and replace with 10 ml *freshly made* BEPS buffer (use more if more than one membrane is used).Place bottle in hybrid oven for 10 min. Make sure a second bottle is used to counterbalance the first. 9. Denature probe in boiling water for 5 minutes, rapidly chill on ice. 10. Remove BEPS buffer from hybrid oven bottle. 11. Add the probe to 10 ml of BEPS buffer and then pour into oven bottle. Incubate at 65!C for 16-72 hr.(16 hr. overnight incubation is usually enough) DAY 2: Perform next morning. 12. Remove probe mix by pouring into 50 ml conical tube. Keep in freezer or discard in radioactive liquid waste container under hood. 13. Wash membrane with WSI in following sequence, monitoring with Geiger counter after each wash: 100 ml for 5 min. (discard in radioactive liquid waste) 100 ml for 10 min. (discard in sink, let water run) 100 ml for 10 min. (discard in sink, let water run) 14. For increased stringency (to decrease background) wash with WSII: 100 ml for 20 min. 100 ml for 20 min. 100 ml for 20 min. 15. Check membrane with Geiger counter. If background radioactivity is acceptable, allow filter to air dry between two 3MM whatman filters (10-30 min.). 16. Wrap membrane in Saran Wrap, develop in cassette with intensifying screen (white) in -70!C freezer from 4 hr. to overnight. BEPS buffer: (25 ml) 1-1.5% non-fat dry milk (0.25-0.375 g) 1mm EDTA (50 microl of 0.5 m stock) 0.5 m NaH2PO4 pH 7.2 12.5 ml 1 m stock) 7% SDS (1.75 g) WSI: (300 ml) 0.5% non-fat dry milk (1.5 g) 1 mm EDTA (600 microl of 0.5 m stock) 40 mm NaH2PO4 pH 7.2 (12 ml 1 m stock) 5% SDS (15 g) WSII: (300 ml) 1 mm EDTA (600 microl of 0.5 m stock) 20 mm NaH2PO4 pH 7.2 (6 ml 1 m stock) 1% SDS (3.0 g) For each of these solutions, dissolve with magnetic stirring at 65 deg C. Removing Probes from Nylon Membranes 1. Immerse the membrane in 100 ml 50% formamide, 2X SSPE for 1-4 hr at 65!C. 2. Rinse the membrane briefly with 100 ml 0.1X SSPE at room temperature. Remove liquid from the membrane by placing on a stack of paper towels. 3. Sandwich damp membrane with Saran Wrap, and monitor with Geiger counter or expose to X-ray film to check that probe has been removed. 4. Membrane can now be dried and stored at room temperature. 20X SSPE: NaCl 175.3 g NaH2PO4aH2O 27.6 g EDTA 7.4 g Dissolve in 800 ml dH2O, adjust pH to 7.4 with ~6.5 ml 10 n NaOH. Adjust volume to 1 liter, sterilize by autoclaving.Explanation & notes: From Maniatis, pp 1.25-1.26
Submitted by: Floriza Mangahas
E-mail address: manga006@maroon.tc.umn.edu
Submission Date:
