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

 Prehybridization solution

                                                                                               Final

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

Prehybridize at 65^OC 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^OC.

 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 (1ml) of template DNA into a microcentrifuge tube. Add in 5ml of primers and appropriate volume of 50ml in the final reaction. Denature by heating to 95-100^OC 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 10ml and enzyme 2ml. Add in 2.5ml 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^OC for 10 minutes
5. Stop the reaction by the addition of 2ml 0.5% EDTA. For use in a hybridization, denature the labelled DNA by heating to 95-100^OC for 5 min, then chilled on ice.
6. Add 50ml probes to each hybridization tube
7. Hybridize o/n at 65^OC

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^OC

W1 = 0.1SSC – 0.1% SDS ® 250ml                      kept at 65^OC

1. Drain the solution from the hybridization tube into a 50ml centrifuge tube. The probe can be stored frozen for re-use.
2. For room temerperature wash, add in W1. Mix it up and throw W1 solution away in the sink. Repeat twice.
3. Add W2. Leave rotating in the wash at 65^OC for 30 min. Repeat.
4. Add W3. Leave the wash at 65^OC for 30 min. Repeat.
5. Take out the membrane and dry it on paper towel. Wrap with Saran film & place it in a film cassette. Monitor radioactivity of the membrane to estimate exposure times: >1000 counts: 2 hours; 200-300 counts, 24 hours. Put x-ray film over on both sides of the membrane under a safelight, place cassette in -20 Freezer (up to 7 days) or -70C freezer, and then develop films.

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 -20^OC

Preparation of 50% dextran sulphate (10ml)

5g Dextran sulphate

10ml autoclaved water

Stir for about 8 hours, heating to 65^OC. 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

www.molcyt.com

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): 

1. Prepare 0.3 M Sodium-phosphate buffer by adding 40.2g NaH₂PO₄ to 400 ml sterile distilled water.
2. Add monobasic sodium phosphate salt gradually to adjust pH to 7.2 while warming up the solution on a hot plate.
3. Add gradually 35g SDS, 10g BSA and 0.185g EDTA.
4. Finally top up buffer to 500 ml.
5. Aliquot 12.5 ml/polypropylene tube and store at room temperature or –20^oC freezer.
6. Prior to use, thaw tubes in  60^oC oven or in running hot tap water to redissolve SDS and BSA. Shake tube intermittently.

 For prehybridization:

1. Dispense the fully dissolved solution into membrane containing Hybaid tube.
2. Rotate tube for 4 to 5 hours at 60^oC or 65^oC before adding the denatured radioactively labelled probe.
3. Continue with hybridization overnight with the same solution and temperature.

Note: The prehyb- and hybridization solution is the same.

          60^oC for lower stringency Southern hybridization.

          65^oC for higher stringency Southern hybridization.

 For probe labelling:

 Use RadPrime DNA Labeling System (Invitrogen) 

1. Denature 25 ng (1 µl) in 19 µl of sterile distilled water or TE in a microcentrifuge tube by heating for 5 min in a boiling water bath; then immediately cooled on ice.
2. Perform the following adddtions on ice:

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

3. Mix briefly.
4. Pipette up and down a tip of  alpha-³²P with the above mixed solution until the reddish  color on pipette tip disappears.
5. Add 1 µl Klenow fragment, centrifuge briefly.
6. Incubate at 37^oC for 4 hrs or until it is ready to be added into the prehybridization solution.
7. Denature probe at 90 - 100^oC for 10 minutes then add immediately into the solution in Hybaid tube.
8. Continue with hybridization overnight.

 Washing of membranes:

1. After overnight hybridization, drain the radioactive solutions into a 15 ml polypropylene tube and store it in the –20^oC freezer for reuse.
2. If hybridization  were done at 60^oC,

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 60^oC for 30’ by rotating in the hybridization oven.

3. If  hybridization were done at 65^oC,

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 65^oC.

iii.     Once with ~12.5 mls 1xSSC/0.1% SDS for 15’ at 65^oC.

         Note: Washings are done in the Hybaid tube itself.

Preparations of washing solutions:

        2xSSC/0.1%SDS:

        5 ml 20xSSC, 0.25 ml 20%SDS, 44.75 ml distilled water.

        1xSSC/0.1%SDS:

        2.5 ml 20xSSC, 0.25 ml 20%SDS, 47.25 ml distilled water.

 Autoradiography:

1. After final wash, remove membrane from tube, blot dry with paper towel and wrap with Saran cling film.
2. Monitor radioactivity of the membrane to estimate exposure times.
3. Place the membrane in a film cassette and put X-ray film on both sides of the membrane. This is to be done in the dark room.
4. Store cassette in –20^oC freezer overnight (500 counts/sec) or a week (10 counts/sec) depending on the radioactivity counts.
5. When ready, take out cassette, thaw to room temperature and then develop film.