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This week’s lab focuses on PCR of environmental isolates, where students will perform a series of experiments to identify potential new bioluminescent species. Students will learn to extract chromosomal DNA, amplify the luxA gene, and analyze the PCR products via agarose gel electrophoresis. Additionally, the purification of PCR products and database sequencing will be undertaken to determine if isolates are known or novel species. Lab reports for labs 12, 13, and 14 are due by March 7th, with questions provided in the handout on the potential identification of new 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
