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

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
    • B + C with IgAb-F and Mut_Pst1b-R
    • C + D with Mut_Pst1a-F and IgAb-R
  • 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