- Electrophoresis of DNA is carried out in a neutral agarose gel system.
Prepare a 0.8%–1% agarose gel containing 1X TAE buffer. Ethidium bromide
can be added to a final concentration of 0.2 mg/mL.
- Apply the samples to the gel.
- Run the gel in 1X TAE buffer at 4V/cm until the bromophenol blue indicates
that the sample has run for a sufficient distance.
- Following electrophoresis, visualize the gel under UV transillumination
and photograph it along with a ruler.
- (i) Depurination—10 minutes at room temperature with gentle agitation
(optional). This step is necessary if target sequences are greater than
10 Kb in size.
(ii) Denaturation—25 minutes at room temperature with gentle agitation.
(iii) Neutralization—30 minutes at room temperature with gentle agitation.
When using nitrocellulose membranes, the neutralization time should be
extended to 45 minutes. Include a rinse in distilled water between each step.
- Assemble the capillary blotting apparatus using 10X SSC as the transfer
buffer. Allow the DNA to transfer overnight onto Hybond N+.
- The following day, disassemble the apparatus, mark the membrane
appropriately and fix the DNA to the membrane by UV cross link orbaking (2 hours at 80°;C). For nitrocellulose membranes, bake for 2 hrs,
at 80°;C in a vacuum oven.
- Hybridization buffer
- 5X SSC
- 1 in 20 dilution liquid block (Amersham) or other blocking reagent
- 0.1% (w/v) SDS
- 5% (w/v) dextran sulfate
- EDTA stock:0.5M EDTA pH8.0
- SDS stock:10% or 20% (w/v) SDS
- Depurination solution (for Southern blotting)
- 250 mM HCl
- Denaturation solution (for Southern blotting)
- 1.5M NaCl
- 0.5M NaOH
- Neutralization solution (for Southern blotting)
- 1.5M NaCl
- 0.5M Tris-HCl
- pH adjusted to 7.5
- 20X SSC:0.3M Na (3) citrate, 3M NaCl