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

Cellular Adhesion

• Oligos
  • IgAbfull-F
  • Resuspended at 50uM

• 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

• 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