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

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