User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/02

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Cellular Adhesion

  • PCR Purification
    • Used MinElute columns to PCR purify the six samples from yesterday (A-F)
    • Eluted in 10ul of water
  • Spec
    • B(IgA-bc Part 1): 104.5 ng/ul
    • C(IgA-bc Part 2): 76.3 ng/ul
    • D(IgA-bc Part 3): 87.1 ng/ul
  • Digest
    • Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
      • B + C + D
      • B + C
      • C + D
    • Thermocycler protocol: 1hr@37C, 20mins@65C
  • Ligation
    • Performed on digest tubes above
    • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
    • Thermocycler protocol: 1hr@16C,10mins@65C
  • Liquid Cultures
    • Inoculated 8ml of LB Tet with Fos containing plasmid
    • Inoculated 8ml of LB Tet with JunB containing plasmid
    • Inoculated 8ml of LB Kan with BBa_C2001
    • Grew up overnight at 37C
  • PCR Purification
    • Used MinElute columns to PCR purify B + C and C + D
    • Eluted in 10ul of water
B + C + D (892 bp), B + C + D (892 bp), log-2 ladder
  • Gel
    • Ran 1% gel for 40 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • Bands might be in the right place (a little short, but hard to tell)
    • Cut and purified band that was near the correct region
  • Spec
    • B + C: 192 ng/ul
    • C + D: 218 ng/ul
  • Digest
    • B + C with D: 3ul D DNA, 1.5ul B + C DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • C + D with B: 3ul B DNA, 1.5ul C + D DNA, 2ul NEB4, .5ul Sap1, rest water (20ul rxn)
    • Thermocycler protocol: 1hr@37C, 20mins@65C
  • Gel Purification
    • QIAEX gel purification
    • Eluted in 20ul of water
  • Ligation
    • Performed on digest tubes above
    • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
    • Thermocycler protocol: overnight@16C,10mins@65C