Southern Blotting

From ActinoBase

There are many different protocols for Southern blotting. This approach uses vacuum transfer pf DNA to a nylon membrane, DIG-labelling of the probe and detection by chromogenic substrate or chemiluminescence. The buffers for DNA transfer are as described by Sambrook et al., transfer is carried out by following the instructions for the vacuum pump. The protocols for labelling and detection are as described by the manufacturer’s instructions (Roche Life science in this case).

Consumables and Solutions

Consumables for Southern Blotting

Solutions for Southern Blotting

Instructions

Labelling the Probe

If a blot fails completely, it will almost always be because of a bad probe. For best results: label around 1 µg DNA. Two options for DIG-labelling are described here:

  1. Random primed DNA labelling: To label a whole cosmid or a fragment of DNA (e.g. fragment excised from a plasmid or a PCR product).You will need a very concentrated DNA sample for this, as each reaction will only label up to 15 µl DNA, and you want approximately 1 µg DNA.
  1. PCR labelling: Labels during PCR and therefore generates a lot of labelled DNA from small amount of template. This is a more expensive method but, provided PCR has already been optimised, is faster and more likely to generate the required amount of probe.

Random Primed DNA Labelling

In this method, DNA is made single stranded and then hexanucleotides act as random primers for second strand synthesis using Klenow enzyme. The DIG DNA labelling mix contains dATP. dCTP, dGTP and DIG-labelled dUTP, so DIG is incorporated into the second strand every 20-25 nucleotides as it is synthesised.

  1. Make volume of DNA sample to 15 µl with water (10 ng – 3µg).
  2. Boil at 95°C for 10 min to denature DNA (can use a PCR machine for best results), then chill quickly in an ice water bath for around 10 min.
  3. Set up the following reaction:
    1. 15 µl denatured DNA
    2. 2 µl Hexanucleotide mix 10x
    3. 2 µl DIG DNA labelling mix
    4. 1 µl Klenow enzyme
  4. Mix and incubate at 37°C for 1h-20h (for best results, leave overnight)
  5. Stop the reaction by adding 2 µl 0.2M EDTA (pH 8.0).
  6. Store at -20°C until required.

PCR Labelling

The PCR DIG labelling mix contains dATP, dCTP, dGTP and DIG-labelled dUTP, so as the DNA is amplified in a standard PCR reaction DIG is incorporated into every product.

  1. Set up PCR reaction as usual but instead of using standard dNTPs use PCR DIG labelling mix. Mix is 10x concentrated so use 5 µl per 50 µl reaction.
  2. Labelled PCR products can be used directly as probe. However, if there are any non-specific products, the correct size product should be excised from an agarose gel and eluted before use.
  3. You can combine the products of several reactions to use as your probe.
  4. Store at -20°C until required.

Electrophoresis

Transfer to Membrane

Hybridisation

Stringency Washes

Detection