* Boiled for 10 min then cooled on ice. Then added to the solution at 65℃.

Prehybridize at 65 O C for 2 to 8 hours, then pour of prehybridization solution; it can be stored at 4C and reused over a week or so.

Hybridization solution Final

* Boiled for 10 min then cooled on ice. Then add to the solution at 65℃.

Add hybridization solution of membranes and rotated for 30 min to 4 hr before addition of labelled probe. The hybridization solution is identical to the pre-hybridization solution except for the inclusion of dextran sulphate. This polysaccharide increases the effective probe concentration in the hybridization solution by displacing probe from the volume occupied by the polymer.

DNA Labelling procedures

Follow a protocol like this or use a kit - BioLine HYPER-Prime, GibcoBRL (and previously Roche but we find their products do not have the reproducibility and robustness of the former Boehringer) are used in our laboratory.

1. Place 25ng (1 m l) of template DNA into a microcentrifuge tube. Add in 5 m l of primers and appropriate volume of 50 m l in the final reaction. Denature by heating to 95-100 ℃ for 5 min in a boiling waterbath
2. Spin briefly in a microcentrifuge to bring the contents to the bottom of the tube. Straight away put on ice.
3. Keeping at room temperature, add in labelling buffers 10 m l and enzyme 2 m l. Add in 2.5 m l radiolabelled (dCTP). Mix gently by pipetting up and down and cap the tube. Spin for a few seconds in a microcentrifuge to bring the contents to the bottom of the tube.
4. Incubate at 37 O C for 10 minutes
5. Stop the reaction by the addition of 2 m l 0.5% EDTA. For use in a hybridization, denature the labelled DNA by heating to 95-100 ℃ for 5 min, then chilled on ice.
6. Add 50 m l probes to each hybridization tube
7. Hybridize o/n at 65 ℃

Filter washing: preparation of washing solution

W1 = 2SSC ?0.5% SDS —> 150ml kept at RT
W1 = 0.5SSC ?0.1% SDS —> 250ml kept at 65 ℃
W1 = 0.1SSC ?0.1% SDS —> 250ml kept at 65 ℃

• Drain the solution from the hybridization tube into a 50ml centrifuge tube. The probe can be stored frozen for re-use.
• For room temerperature wash, add in W1. Mix it up and throw W1 solution away in the sink. Repeat twice.
• Add W2. Leave rotating in the wash at 65℃ for 30 min. Repeat.
• Add W3. Leave the wash at 65℃ for 30 min. Repeat.
• Take out the membrane and dry it on paper towel. Wrap with Saran film
  

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Southern Hybridization Protocols
点击：   作者：51protocol收集   来源：  时间： 2007-08-30 　本站论坛
Two protocols are given here. The first one is used for BAC library screening on filters , although is also suitable for Transfer hybridizations. PREPARATION OF HYBRIDIZATION SOLUTION Prehybridization solution Final
dH2O 1M Tris pH 8.0 0.5M EDTA pH 8.0 20X SSC 50X Denhardts 20% SDS *SS DNA 10 mg/ml	16.00 ml 1.25 ml 0.50 ml 6.25 ml 0.50 ml 0.25 ml 0.25 ml 25.00 ml	- 50 mM Tris 10mM EDTA 5 x SSC 1 x Denhardts 0.2 SDS 100 m g/ml
dH2O 50% dextran sulfate 1M Tris pH 8.0 0.5M EDTA pH 8.0 20X SSC 50X Denhardts 20% SDS *SS DNA 10 mg/ml	11.00 ml 5.00 ml 1.25 ml 0.50 ml 6.25 ml 0.50 ml 0.25 ml 0.25 ml 25.00 ml	- 10% dextran sulfate 50 mM Tris 10mM EDTA 5 x SSC 1 x Denhardts 0.2 SDS 100 m g/ml
W1	W2	W3
15ml 20XSSC	6.25ml 20XSSC	1.25ml 20XSSC
3.75ml 20% SDS	1.25ml 20% SDS	1.25ml 20% SDS
131.25ml H2O	242.5ml H2O	247.5ml H2O
150.00ml	250.00ml	250.00ml