Southern Blotting

Question 1

Five main stages

Answer 1

Preparation of restriction enzyme;

Gel electrophoresis;

Blotting;

Hybridization w radioactively labeled probes;

Auto radiology

Question 2

Details of preparing restriction fragments

Answer 2

Digestion of DNA samples w same restriction endonuclease

Question 3

Details of blotting

Answer 3

Immersed in NaOH buffer to denature duplex DNA into ss;

Gel is overlaid w a nitrocellulose membrane;

Thick wad of paper toweling is stacked above acting as a wick;

Drawing up the buffer sol through the filter by capillary action;

Transferring ssDNA onto nitrocellulose membrane;

Nitrocellulose paper bonds ssDNA v tightly

Question 4

Describe hybridization w radioactively labeled probes

Answer 4

DNA probe consists of ssDNA complementary to the target DNA seq;

Probe bonds to any restriction fragments containing part of target DNA seq;

Unbound probe washes away

Question 5

Describe auto radiography

Answer 5

X-Ray film is laid over the nitrocellulose blot;

Position of DNA seq that form base pairs w the probe are revealed as a dark band on the film