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Overview. Wednesday PCR of environmental isolates Discuss the restriction analysis gels Thursday Run gel of environmental isolate PCR PCR purification Handout Labs 12, 13 & 14 due March 7 th Questions in handout. A new bioluminescent species?.
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Overview • Wednesday • PCR of environmental isolates • Discuss the restriction analysis gels • Thursday • Run gel of environmental isolate PCR • PCR purification • Handout • Labs 12, 13 & 14 due March 7th • Questions in handout
A new bioluminescent species? • You will attempt to identify your environmental isolates • Extract chromosomal DNA • Amplify a fragment of the luxA gene • Sequence the PCR product • Perform a database search with the resulting sequence • Determine whether the isolate is a known species or a novel one
Wednesday • Boiling lysis to prep environmental samples for PCR • handout • Set up environmental isolate PCR • 3 reactions with chromosomal DNA dilutions • 2 negative controls (1 no primers, 1 no template) • 1 positive control (5ng pUW500 plasmid) • Each tube should contain the following: • 10 ul 2X PCR mix • 2 ul forward primer • 2 ul reverse primer • 1 ul DNA (1:10, 1:100, 1:1000) • 5 ul water
Checklist Each person sets up PCR of their own isolates • 1:1, 1:10, 1:100 dilutions of template to be used • one positive control • 2 negative controls
Thursday • Agarose gel analysis of PCRs from environmental isolates • Pour gel as before (80 ml, 1% agarose) • Use two combs • Add 5 ul of loading dye to PCRs, load 10 ul • Stain and image • PCR clean-up using spin columns • Handout
