User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/19

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Cellular Adhesion

  • Liquid Culture
    • Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from plate colonies
    • Inoculated 8ml Amp/Kan LB with autotransporter from plate colonies
    • Put in incubator at 12:15 AM
  • Colony PCR
    • Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
    • Picked three colonies from each plated sample to PCR
      • B+C w/ D (IgA primers)
      • C+D w/ B (IgA primers)
      • B+C w/ D (BB primers)
      • C+D w/ B (BB primers)
      • 6His Tag
      • FLAG Tag
      • Myc Tag
      • HA Tag
      • GCN4 Leucine Zipper
    • Grew up liquid cultures of all samples for miniprepping (if gel shows correct insert)
    • Protocol:
      • 95C for 15 mins
      • 94C for 30 secs
      • 55C for 30 secs
      • 68C for 1.5 min
      • REPEAT 2-4 39 times
      • 68C for 10 mins
      • 4C FOREVER
  • Gel
    • Ran 1.5% for 30 minutes at 100V to see if PCR product is correct length (5ul of PCR product with 2ul of dye)
      • B+C w/ D (IgA primers): 2 unique (none look right)
      • C+D w/ B (IgA primers): 2 unique (none look right)
      • B+C w/ D (BB primers): 2 unique (none look right, contains longest fragment of all autotransporters)
      • C+D w/ B (BB primers): 2 unique (none look right)
      • 6His Tag: 3 unique (sequence all)
      • FLAG Tag: 3 unique (sequence all)
      • Myc Tag: 2 unique (sequence both)
      • HA Tag: 2 unique (sequence both)
      • GCN4 Leucine Zipper: 1 unique (sequence)
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