Southern blotting & hybridization
Transfer | Hybridization | Uses | Variations
- In Southern blotting, denatured DNA is transferred from an electrophoresis gel to a sheet of nitrocellulose or derivatized nylon.
- The exploded view at right shows that transfer is accomplished by the flow of buffer through wick, gel, sheet and into adsorbent paper layers.
- DNA is retained by the derivatized nylon or nitrocellulose. After transfer, the DNA is permanently attached by heating or by cross-linking using UV radiation.
- Spatial arrangement of DNA in the gel is preserved in the sheet.
- In hybridization, the sheet with attached DNA can be incubated with a tagged denatured DNA population (probe).
- If probe molecules are complementary to molecules bound on the sheet, double-stranded DNAs will form, thus binding the probe to the sheet.
- After excess probe and poorly bound probe molecules are removed by washing, the only tagged molecules left are those that have formed hybrids with the target DNA.
- Dot and slot blots, in which the sample is spotted directly on the nylon or nitrocellulose sheet without prior separation.
- Northern blots in which the molecules separated by electrophoresis are RNAs instead of DNAs.
- Colony lifts, in which the pattern of colonies on an agar plate is reproduced as a pattern of DNA spots on a nitrocellulose or nylon sheet.
- Plaque lifts, similar to colony lifts except the material lifted is from a phage plating.
- Further information:
- A "Shockwave" animation is available no longer (let U. Melcher know if you know where to find it!).
Plasmid analysis is illustrated in a You tube video
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This is page 423 of Molecular Genetics by Ulrich Melcher, ©1997, 1998, 2014
E-mail inquiries to U. Melcher------------Last Updated: 30 August, 2014