User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/12

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Cellular Adhesion

  • Glycerol
    • Took 1.6ml of liquid culture and created 10% glycerol stocks
      • IgAss + Fos #1
      • IgAss + Fos #2
      • IgAss + Fos #3
      • IgAss + JunB #1
      • IgAss + JunB #2
      • IgAss + JunB #3
      • GCN4 #4
      • GCN4 #5
      • GCN4 #6
  • Miniprep
    • Extracted DNA from remaining liquid culture (~6ml)
    • Eluted in 50ul water
  • Digest
    • Template (20ul rxns): 3ul DNA, 2ul NEB4, .5ul Sap1, rest water
      • B: Part 1 of IgAbc
      • C: Part 2 of IgAbc
      • D: Part 3 of IgAbc
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Performed on today's digest tubes
    • Template (50ul rxns): 10ul of each digest, 5ul ligase buffer, .5ul ligase, rest water
      • B + C
      • C + D
    • Thermocycler protocol: 1hr@16C,15mins@65C
  • PCR
    • Template: 40ul PCR Supermix, .4ul of each primer, and 1ul of ligation product
    • Same thermocycler protocol as 8.1.2007
B + C, C + D, log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • Everything looks fine
  • Digest
    • B + C with D: 1.5ul D digest, 1.5ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • C + D with B: 1.5ul B digest, 1.5ul C + D DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Template (50ul rxns): 20ul of each digest, 8.5ul of remaining digested DNA(B and D), 5ul ligase buffer, .5ul ligase, rest water
      • B + C with D
      • C + D with B
    • Thermocycler protocol: 1hr@16C,10mins@65C
  • PCR
    • Conservative IgAbc F (B + C with D): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
    • Conservative IgAbc R (C + D with B): 40ul PCR Supermix, .4ul IgAb-F and IgAb-R, 1ul ligation product
    • Same thermocycler protocol as 8.1.2007