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

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