User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/17

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Cellular Adhesion

log2-ladder, B, C, D, B + C, C + D, B + C + D (IgA primers), B + C + D (BB primers), Complete IgAbc without muts, log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • Repeat of last gel yesterday, but with more fragments of known length
    • Same conclusion as last gel of 8.16.2007
  • PCR Purification
    • Used MinElute columns to PCR purify tubes B+C, C+D
    • Eluted in 10ul of water
  • Digest
    • Template (20ul rxns): 3ul DNA, 2ul NEB2, .2ul BSA, .5ul EcoR1, .5ul Pst1, rest water
      • 6His Tag
      • FLAG Tag
      • Myc Tag
      • HA Tag
      • GCN4 Leucine Zipper
    • Thermocycler protocol: 1hr@37C, 20mins@80C
    • Note: DNA was not PCR purified (it shouldn't matter)
  • Digest
    • Template: 3ul DNA (each), 2ul NEB2, .5ul Ear1, rest water (20ul rxn)
      • B+C with D
      • C+D with B
    • Thermocycler protocol: 1hr@37C, 20mins@80C
  • Ligation
    • Template: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
    • Ligated all digested samples from the first digest of the day (antibody tags and GCN4 leucine zipper)
    • Thermocycler protocol: 30mins@roomtemp,10mins@65C
  • Ligation
    • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
    • Ligated all digested samples from the second digest of the day (IgAbc samples)
    • Thermocycler protocol: 30mins@roomtemp,10mins@65C
  • Transformation
    • 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
    • 45secs heat shock at 42C using water bath
    • Added 300ul of SOC
    • Incubated for 1hr at 37C
    • Plated on Amp/Cl and grew up at 37C overnight
  • PCR
    • Template: 40ul PCR Supermix, .4ul of each IgA primer (.8ul of each BB primer), and 1ul of ligation product
      • B+C with D
      • C+D with B
      • Both samples PCRed with both IgA primers and BB primers
    • Protocol same as 8.1.2007
B+C with D (IgA primers), B+C with D (BB primers), C+D with B (IgA primers), C+D with B (BB primers), log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (5ul sample with 2ul of loading dye)
    • BANDS ARE AT THE CORRECT LENGTH!!! WE HAVE COMPLETE IGA BETA-CORE WITH MUTATIONS!!!