VGEC: Teacher/Student Notes Southern Blot Protocol

MATERIALS 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 NB steps 1 5 and 13 are NOT shown on the video 1. 2. 3. 4. 5. 6. 7. 8. 9. Place the gel on the small glass plate into a small tray and cover with water to rinse. Remove the water using an aspirator connected to a tap by a sink. Cover the gel with depurinating solution and shake gently for 10 mins. Remove the solution and repeat. Remove the second lot of depurinating solution and cover the gel in denaturing solution. Shake for 10 mins., remove and repeat. After removing the second lot of depurinating solution, add the neutralising solution and, in the same manner as steps 3 and 4 above, carry out two washes, each of 10 mins. Carefully and wearing gloves, place the 3 small pieces of 3MM paper and then the Hybond filter into a tray containing 2 x SSC. Soak several pieces of the 3MM filter paper wick in 20 x SSC and place in a clean tray. Carefully place the gel on the filter paper, avoiding trapping air bubbles under the gel. 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 the last piece of clingfilm.

10. Put a small amount of 20 x SSC on the gel, and then remove the Hybond filter from the 2 x SSC and carefully place it onto of the gel, avoiding trapping air bubbles. Virtual Genetics Education Centre: http://www.le.ac.uk/ge/genie/vgec/

11. Place the soaked 3MM filter paper onto of the gel and if needed, top up with another small amount of 20 x SSC to keep the filter paper damp. Gently roll over the 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 weight on top. Leave overnight. 13. The following day, the filters should be exposed to UV light to bind the DNA to the filter. They are then ready to be hybridised and probed before being exposed to x-ray film.

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