User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/24

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Cellular Adhesion

  • 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 (five tags and GCN4)
    • Picked a large, medium, and small colony (in that order)
    • Some plates only had three colonies
  • Liquid Cultures
    • Inoculated 8ml Amp/Cl LB with antibody tags and GCN4 from yesterday's plate colonies
    • Grew up in 37C overnight
  • Gel
    • Ran 1% gel for 30 minutes at 95V with samples (5ul sample with 2ul of loading dye)
    • Again, poor resolution with 1% gel, but relative lengths look good
log2-ladder, NEW B, NEW C, NEW D
  • PCR Purification
    • Used MinElute columns to purify the NEW B, NEW C, NEW D from 1.23 PCR
    • Eluted in 10ul of water
  • Digest
    • Template for IGA (single): 3ul DNA (each), 2ul NEB2, .5ul Sap1, rest water (20ul rxn)
      • NEW B + NEW C
      • NEW C + NEW D
      • NEW B + NEW C + NEW D
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • 6ul of IGA digest DNA, 7ul water, 1.5ul ligase buffer, .5ul ligase
    • Protocol: 30mins@roomtemp,10mins@65C