1. 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

2. 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

3. 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

4. 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

5. 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