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

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)