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

Cellular Adhesion

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

• 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 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