User:Mbennie/Notebook/Lab Notebook/Notebook/2007/08/14

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Cellular Adhesion

B+C with D, Complete IgAbc without muts, 2-log ladder
B+C with D, Complete IgAbc without muts, 2-log ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (20ul sample with 5ul of loading dye)
    • There is no band at the correct length
    • Problem might be caused by PCR: primer is now binding to entire piece of DNA instead of just half of it
  • PCR
    • Template: 20ul PCR Supermix, .2ul VF2 and VR, .5ul miniprep DNA
      • SS+Fos #1
      • SS+Fos #2
      • SS+Fos #3
      • SS+JunB #1
      • SS+JunB #2
      • SS+JunB #3
    • Protocol: Same as 8.1.2007
  • Gradient PCR
    • Template: 10ul PCR Supermix, .2ul BB_f and BB_r, .5ul ligation DNA (B+C with D)
      • Ran eight samples from 55-70C on gradient PCR (other than annealing temp, everything else is the same as 8.1.2007 protocol)
    • Template: 10ul PCR Supermix, .1ul IgAb-F and IgAb-R, .5ul ligation DNA (B+C with D)
      • Ran eight samples from 55-70C on gradient PCR (other than annealing temp, everything else is the same as 8.1.2007 protocol)
SS+Fos #1, SS+Fos #2, SS+Fos #3, SS+JunB #1, SS+JunB #2, SS+JunB #3, log-2 ladder
SS+Fos #1, SS+Fos #2, SS+Fos #3, SS+JunB #1, SS+JunB #2, SS+JunB #3, log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (10ul sample with 4ul of loading dye)
    • Looks like everything worked except SS+JunB #2 (~300bp)
Gradient PCR from 55 to 70 with BB primers, Complete IgAbc without muts (mixed last two samples up), log-2 ladder
Gradient PCR from 55 to 70 with BB primers, Complete IgAbc without muts (mixed last two samples up), log-2 ladder
Gradient PCR from 55 to 70 with IgAb primers, Complete IgAbc without muts, log-2 ladder
Gradient PCR from 55 to 70 with IgAb primers, Complete IgAbc without muts, log-2 ladder
  • Gel
    • Ran 1.5% gel for 30 minutes at 100V with samples (10ul sample with 4ul of loading dye)
    • All bands are at 300bp
    • WHAT IS THIS?!?!
    • Possible Solution: By not PCR purifying tube D, it amplified D instead of the entire fragment
  • PCR
    • Template: 40ul PCR Supermix, .4ul VF2 and VR, .8ul DNA
      • PCRed tube D to make some more
    • Protocol: Same as 8.1.2007
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