These pages are the sole responsibility of Pat Heslop-Harrison.
Department of Biology, University of Leicester, LE1 7RH UK
Phone: +44/0 116 252 5079 / 3381
FAX: +44/0 116 252 2791
Southern Hybridization Protocols
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
* Boiled for 10 min then cooled on ice. Then added to the solution at 65OC.
Prehybridize at 65OC 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 65OC.
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.
Filter washing: preparation of washing solution
W1 = 2SSC – 0.5% SDS ® 150ml kept at RT
W1 = 0.5SSC – 0.1% SDS ® 250ml kept at 65OC
W1 = 0.1SSC – 0.1% SDS ® 250ml kept at 65OC
Preparation of Denhardts Solution (30ml)
300mg Ficoll 400 (Fisher Bioreagents)
300mg PVP (Polyvinyl pyrrolidone) (Fisher Bioreagents)
300mg BSA (A-9647 Sigma)
Top up autoclaved H2O up to 30ml (do NOT autoclave solution: BSA will coagulate!)
Filter with 0.2 m. Store at -20OC
Preparation of 50% dextran sulphate (10ml)
5g Dextran sulphate
10ml autoclaved water
Stir for about 8 hours, heating to 65OC. Force through an 0.22 um filter (very hard work!) to sterilise and dispense to to aliquots which are stored at -20C. Do not autoclave!
Pat Heslop-Harrison, Karine Alix, Saadiah Jamli
University of Leicester LE1 7RH
phh4 @ le.ac.uk
Protocol 2. From Sybille Kubis, Maria Madon and Pat Heslop-Harrison.
SOUTHERN HYBRIDIZATION PROTOCOL
Prepare prehyb- and hybridization solution which consists of the following components:
0.3 M Sodium-phosphate buffer pH 7.2
7% (w/v) SDS
2% (w/v) BSA
1 mM EDTA pH 8.0
Preparation of solution (not easy to prepare therefore follow the recipe):
Note: The prehyb- and hybridization solution is the same.
60oC for lower stringency Southern hybridization.
65oC for higher stringency Southern hybridization.
For probe labelling:
Use RadPrime DNA Labeling System (Invitrogen)
1 µl 500 µM dATP
1 µl 500 µM dGTP
1 µl 500 µM dTTP
20 µl 2.5x Random Primers Solution
7 µl distilled water
Washing of membranes:
rinse membranes :-
i. Once with ~12.5 mls 2xSSC/0.1% SDS at room temperature. Drain the solution carefully into the sink outlet.
ii. Then twice with ~12.5 mls 2xSSC/0.1%SDS at 60oC for 30’ by rotating in the hybridization oven.
rinse membranes :-
i. Once with ~12.5 mls 2xSSC/0.1%SDS at room temperature. Drain the solution carefully into the sink outlet.
ii. Twice with ~12.5 mls 2xSSC/0.1%SDS for 30’ at 65oC.
iii. Once with ~12.5 mls 1xSSC/0.1% SDS for 15’ at 65oC.
Note: Washings are done in the Hybaid tube itself.
Preparations of washing solutions:
5 ml 20xSSC, 0.25 ml 20%SDS, 44.75 ml distilled water.
2.5 ml 20xSSC, 0.25 ml 20%SDS, 47.25 ml distilled water.