User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/18

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Cellular Adhesion

  • Solid Culture
    • No colonies -> I guess I should have have PCR purified the samples
    • Actually samples were grown up on Cl/Kan and did not have Kan resistance -> no cells
  • PCR Purification
    • Used MinElute columns to PCR purify autotransporter (4), antibody tags(4), and GCN4 leucine zipper (1)
    • Eluted in 10ul of water
  • Digest
    • Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
      • B+C with D (IgA primers)
      • C+D with B (IgA primers)
      • B+C with D (BB primers)
      • C+D with B (BB primers)
      • 6His Tag
      • FLAG Tag
      • Myc Tag
      • HA Tag
      • GCN4 Leucine Zipper
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Template: .2ul vector (1AC3 for antibody tags and leucine zipper, 1AK3 for autotransporter), 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
    • Ligated all samples from today's digest
    • Thermocycler protocol: 30mins@roomtemp,10mins@65C
  • Transformation
    • 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
    • 45secs heat shock at 42C using water bath
    • Added 300ul of SOC
    • Incubated for 1hr at 37C
    • Plated on Cl or Kan (made some more Cl/Amp and Kan/Amp plates) and grew up at 37C overnight