Overview Wednesday Thursday Labs 12, 13 & 14 due March 7th 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