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 |
* 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
|
dH2O |
11.00 ml |
- |
* 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.
- 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
- Spin briefly in a microcentrifuge to bring the contents to the bottom of the tube. Straight away put on ice.
- 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.
- Incubate at 37 O C for 10 minutes
- 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.
- Add 50 m l probes to each hybridization tube
- Hybridize o/n at 65 ℃
