Week 7
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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.

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Week 7 l.jpg
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


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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


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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!


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Checklist

  • Per table:

    • 5 broth innoculations

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

  • Per Student:

    • 5 PCR in thermocycler


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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


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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


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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


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Next week:

  • Wednesday:

    • PCR of our environmental isolates

    • Ex 12 due with questions

  • Thursday

    • Check PCR with gel and sequence products


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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


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