MICRB320 Lecture Notes - Lecture 4: Proteobacteria, Terminal Restriction Fragment Length Polymorphism, Fluorescent Tag
22 Oct 2015
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Melt it - constant, then create a gradiant with urea and formamide. Temperature gradient - very expensive, so most use denaturing system. Fingerprinting - dgge: denaturing gradient gel electrophoresis (dgge, acrylamide gel with gradient of denaturants, denaturants melt dna, temperature (constant @ 60 c), formamide and urea (gradient, temperature gradient gel electrophoresis (tgge) has higher t (gradient) All the same size, but diff sequences b/c of mixed community. Apply to gel - separate based on melting behaviour. Fingerprinting - dgge: electrophoresis mobilizes the dna through a. Run overnight for better results current: 4-16 hr @ 75-200 volts, longer running time at lower voltage gives better band resolution, a fragment of the 16s rrna gene (300-700 bp) is amplified (pcr) from edna. Pcr amplifies a single sized product of many sequences (edna is a mixed template) Just like human ngerprinting, but alittle diff b/c it"s just melting dna patterns.
