102 handout 1129 - MCB102 Handout 11/29 SOUTHERN BLOTTING 1...

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MCB102 Handout 11/29 SOUTHERN BLOTTING 1. Digest genomic DNA with restriction enzymes 2. Perform agarose gel electrophoresis 3. Denature DNA with base 4. Transfer to a membrane 5. Incubate the membrane with a radioactively labeled probe 6. Wash away unbound probe 7. Expose membrane to film (result is an autoradiogram) Applications of Southern blotting Cloning the DNA fragment that hybridizes to the probe Detect DNA rearrangements, polymorphisms or mutations Restriction fragment length polymorphisms ( RFLPs )-loss or gain of restriction enzyme sites from DNA sequence changes generate different fragment sizes in a Southern blot RFLPs are the basis for DNA fingerprinting -many probes are used to generate Southern blot patterns that, taken together, are unique to a single person DNA SEQUENCING SANGER METHOD 1. Denature double-stranded plasmid template or use single-stranded plasmid template 2. Anneal a large excess of complementary single-stranded primer 3.
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