Blotting

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

 

Southern blotting

A Southern blot is a replica of the restriction pattern formed by DNA fragments in an agarose gel, made on a nylon membrane sheet. Three methods are commonly used to transfer nucleic acids from gels to nylon membranes: capillary, electrophoretic, and vacuum transfer. Capillary transfer does not require any special equipment. It uses the flow of a 0.4M NaOH solution to deposit the DNA fragments on the membrane. A wet sheet of blotting paper acts as a wick for the transfer solution, which is drawn up by a stack of dry paper through the gel/membrane sandwich.

  1. A weigt of about 0.5 kg
  2. A plate, made of glass, PVC or similar.
  3. A stack of dry paper towels
  4. A sheet of pre-wetted nylon blotting membrane with a double layer of pre-wetted gel blotting paper put on top
  5. The gel
  6. The wick: a double layer of pre-wetted gel blotting paper
  7. A plate similar to 2
  8. Two open containers filled with 0.4M NaOH
  9. An supporting empty container, upside down.

for 8 and 9, one large open container with a small plate on top, may be used

 

During the capillary transfer of 0.4M NaOH from containers 8 into the paper towels 3, the DNA is denatured (made single-stranded) by the high pH of the transfer solution, and it is bound to one side of the membrane sheet 4. The binding is covalent when positively charged nylon is used. Uncharged nylon needs to be "baked" at 80 C or treated with UV light, after the blotting step, to make the DNA binding covalent. Small DNA fragments are efficiently transferred in about 1 hour; fragments longer than 15,000 base pairs require an overnight transfer and, even then, may not be completely transferred.
After the blotting using 0.4 M NaOH, the DNA bands have bound to the nylon sheet, the EthBr has been washed out of the gel and into the paper towels, and the gel is flattened to form a very sturdy gel.
If you would like to check the successful transfer of the DNA bands, you should incubate the resulting gel for at least one hour in a large volume of TAE buffer with EthBr. Use a UV lightbox to check for any remaining bands in the gel.

note:
Additional safety precautions (lab coat, safety glasses) should be taken for handling 0.4 M NaOH solutions

 

  See also the Movie about  Blotting

Read the Movie Text