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

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

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!

Checklist

• Per table:– 5 broth innoculations

• Same five innoculations spotted on a plate in the 37C incubator

• Per Student:– 5 PCR in thermocycler

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

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

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

Next week:

• Wednesday:– PCR of our environmental isolates– Ex 12 due with questions

• Thursday– Check PCR with gel and sequence products

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