Week 7

1. Week 7 • Wednesday: • Screening of library transformants • Innoculation of colonies for plasmid preps • Practice PCR • Turn in Lab #11 • Thursday: • Plasmid minipreps • Restriction digestion of minipreps • Run gel of PCR and restriction digest

2. Screening of library transformants • Examine each transformation plate in the dark – at least 10 min • Pick 5 colonies • 1 pUWL500 colony • 4 colonies from your ligation plates • Inoculate the same colony in TSA broth and on an LB amp plate • Place plate in 37C incubator, place liquid in a rack for shaking

3. Practice PCR • Set up PCR to amplify a fragment of the LuxA gene from plasmids • Each student will prepare 5 tubes • 3 dilutions of the plasmid • 1 no template negative control • 1 no primer negative control • See handout for more details!

4. Checklist • Per table: • 5 broth innoculations • Same five innoculations spotted on a plate in the 37C incubator • Per Student: • 5 PCR in thermocycler

5. Thursday: • Plasmid Preps from yesterdays innoculations (Gene Jet protocol) • After plasmid preps are complete, digest with Xba I for 45 minutes • Run gel of preps and PCR products from last time

6. Plasmid minipreps & restriction analysis • Harvest the bacterial culture by centrifugation 1.5 ml at 8000 rpm for 1 min • Decant the supernatant and remove remaining medium • Repeat • Follow the “GeneJet” protocol exactly, starting with step 1 – Cell pellet resuspension • Centrifugation steps should be done at top speed

7. Restriction analysis • Each table will have 5 products. Digest each of these and your plasmid prep from ex 6 • Each tube should contain the following: • 4 ul DNA • 2 ul 10X buffer • 1 ul XbaI • 13 ul water

8. Next week: • Wednesday: • PCR of our environmental isolates • Ex 12 due with questions • Thursday • Check PCR with gel and sequence products

9. Checklist • Plasmid prep of all broths • Digestion of plasmid preps and Ex. 6 palsmid prep • Gel of digestion and PCR from Wed. • Picture of gel