User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/09

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Cellular Adhesion

  • Colony PCR
    • Performed on seven colonies of 8.3.2007 IgAbc-R plate
    • Template: 20ul Supermix, .8ul VF2 and VR, 1 ul of cell dilution (colony in 100ul of water)
    • Plated picked colonies on Amp/Cl plate and grew up at 37C all day and overnight
    • Note: Annealing temp was initially 45C, noticed after 5 cycles, restarted protocol without initial 95C denature and correct annealing temperature
    • Protocol:
      • 95C for 15 mins
      • 94C for 30 secs
      • 55C for 30 secs
      • 68C for 1.5 mins
      • REPEAT 2-4 39 times
      • 68C for 10 mins
      • 4C FOREVER
  • PCR
    • Full Iga beta: IgAbfull-F and IgAb-R
    • Template: 40ul PCR Supermix, .4ul of each primer, and .4ul N. gonorrhea DNA
    • Same protocol as the colony PCR
  • Digest
    • Template (20ul rxns): 3ul DNA (of each), 2ul NEB4, .5ul Sap1, rest water
      • C + D with B
      • B + C
      • C + D
    • Thermocycler protocol: 1hr@37C, 20mins@65C
1-4 IgAbc colonies, 5 IgAbc without muts, 6-8 IgAbc colonies, 9 IgAbfull, 10 log-2 ladder
1-4 IgAbc colonies, 5 IgAbc without muts, 6-8 IgAbc colonies, 9 IgAbfull, 10 log-2 ladder
  • Gel
    • Ran 1.5% for 50 minutes at 100V to see if PCR product is correct length
  • Liquid Culture
    • Grew up cultures in 8ml Amp/Cl LB
      • GCN4 #1 from 8.4 colony PCR backup plate
      • GCN4 #3 from 8.4 colony PCR backup plate
      • IgAbc #8 from 8.9 colony PCR cell dilution
  • Ligation
    • Performed on today's digest tubes
    • Template (50ul rxns): 20ul digest, 5ul ligase buffer, .5ul ligase, rest water
    • Thermocycler protocol: 1hr@16C,10mins@65C
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