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

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