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 1.Plasmid prep of all broths 2.Digestion of plasmid preps and Ex. 6 palsmid prep 3.Gel of digestion and PCR from Wed. 4.Picture of gel