Beginning Molecular Biology Laboratory Manual

 

1. 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 ug/ml.
2. Apply the samples to the gel.
3. Run the gel in 1x TAE. buffer at 4V/cm until the bromophenol blue indicates that the sample has run for a sufficient distance.
4. Following electrophoresis, visualize the gel under UV transillumination and photograph with a ruler.
5. 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
6. Assemble the capillary blotting apparatus using 10X SSC as the transfer buffer. Allow the DNA to transfer overnight onto Hybond N+.
7. The following day, disassemble the apparatus, mark the membrane appropriately and fix the DNA to the membrane by UV crosslinking or baking (2 hours at 80EC). For nitrocellulose membranes, bake for 2 hrs. at 80EC 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 sulphate

EDTA stock

0.5M EDTA pH8.0

SDS stock

10% or 20% (w/v) SDS

Depurination solution (for

Southern blotting)
250mM 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