Nucleic Acid Hybridization Assays
Southern Transfer
The mini gels are 10 x 7 cm with
the wells. You will need:
a. 2-3 cm thick sponge same size or slightly greater than gel
b. 5 pieces of 3 mm chromatography paper 10x7 cm.
c. 3 cm stack of paper towels 12x 9
d. Charged membrane 10 x 7
e. 20 x SSC or 0.4N NaOH
f. if using 20 x SSC you will need depurination solution .25N HCl, Denaturation
solution 1.5M NaCl /.5N NaOH and Neutralization solution 1.5M NaCl/.5M Tris
pH 7.5
High Salt Transfer
1. Slowly rotate the gel in depurination solution until the loading dyes change
color, 15-20 minutes.
(the xylene cyanol changes to yellow green and the bromphenol blue is yellow)
2. Rinse the gel in ddH2O 5 minutes
3. Place in denaturation solution for 30 minutes
4. Rinse the gel in ddH2O 10 minutes
Go to blotting section
Alkaline Transfer
1. Depurinate as above
2. Incubate in .4N NaOH or 8 mN NaOH/3M NaCl (Chomczynski) and go to stack assembly.
Blotting Section
1. Wet the membrane in H2O and then soak in SSC or alkaline
transfer buffer while assembling the stack.
2. Wet three of the five sheets of chromatography paper in transfer solution
3. In a glass dish assemble the paper towel stack and the two dry pieces of
paper.
4. Place the gel upside down on a piece of saran.
5. Bow the membrane and place on the gel; if any air bubbles get caught use
a Pasteur pipet to roll them out.
6. Place one sheet of wet paper on the stack
7. Flip the gel/membrane on top of the stack
8. Mark the wells on the membrane in pencil
9. Cut off the wells or fill with 1% molten agarose.
10. (Optional) place strips of parafilm on edges of gel toprevent short circuiting
of the transfer.
11. Check for any bubbles
12. Soak a sponge in transfer buffer and allow to drain slightly.
(Andy Witherell, last update, Oct 2004)