Blotting Techniques: Gel Electrophoresis

Gel Electrophoresis can separate macromolecules (DNA/RNA, protein) based on their size and charge. Larger molecules move slower through gel matrix), and ones with less negative charge would move slowly towards the positive electrode. The smallest molecules travel furthest but secondary and tertiary structures also affect mobility. Molecules can be denatured which alters secondary structure so only molecular size assessed.

Southern Blotting

• Named after Dr Southern. Southern Blotting allows the visualisation of individual DNA fragments. Double-stranded DNA is cut with restriction enzymes, then undergoes electrophoresis producing a smear based upon size.
• A nylon membrane is placed over the Gel and the DNA transfers. Labelled DNA probes can then be added.
• Once heated the DNA strands come apart and the DNA probes then attach to their complementary sequences. The membrane is washed and then analysed.
• Labelling may be with a radioactive piece of DNA incorporating ³²P which can then be analysed with autoradiography.

Other variations