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Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/

VGEC: Teacher/Student Notes

Southern Blot ProtocolMATERIALS

Distilled water

Depurinating solution (0.25 M HCl)Denaturing solution (0.5 M NaOH, 1.5 M NaCl)

Neutralising solution (0.5 M Tris-HCl, pH 7.4, 3 M NaCl)

20 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M Na citrate)

2 x SSC

Tray and glass plate for washing gel

Blotting apparatus (tray, glass sheet, 3MM filter paper wick, clingfilm)

Hybond-N filter and 3MM filter paper cut to fit gel

Tray for wetting the Hybond and 3MM filters

Paper towels and a weight

BLOTTING THE GEL

NBsteps 15 and 13 are NOTshown on the video

1. Place the gel on the small glass plate into a small tray and cover with water torinse.

2. Remove the water using an aspirator connected to a tap by a sink.

3. Cover the gel with depurinating solution and shake gently for 10 mins. Remove thesolution and repeat.

4. Remove the second lot of depurinating solution and cover the gel in denaturingsolution. Shake for 10 mins., remove and repeat.

5. After removing the second lot of depurinating solution, add the neutralising solutionand, in the same manner as steps 3 and 4 above, carry out two washes, each of10 mins.

6. Carefully and wearing gloves, place the 3 small pieces of 3MM paper and then theHybond filter into a tray containing 2 x SSC.

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Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/

7. Soak several pieces of the 3MM filter paper wick in 20 x SSC and place in a cleantray.

8. Carefully place the gel on the filter paper, avoiding trapping air bubbles under thegel.

9. Surround 3 edges of the gel with clingfilm and if necessary, top up with a small

amount of 20 x SSC to ensure the filter paper remains damp, before adding thelast piece of clingfilm.

10. Put a small amount of 20 x SSC on the gel, and then remove the Hybond filterfrom the 2 x SSC and carefully place it onto of the gel, avoiding trapping airbubbles.

11. Place the soaked 3MM filter paper onto of the gel and if needed, top up withanother small amount of 20 x SSC to keep the filter paper damp. Gently roll overthe top with a glass pipette to ensure no air bubbles are trapped.

12. Add the paper towels on top, then cover with a sheet of glass and place a weighton top. Leave overnight.

13. The following day, the filters should be exposed to UV light to bind the DNA to thefilter. They are then ready to be hybridised and probed before being exposed to x-ray film.