User:Tara K. Luckau/Notebook/Team ConGen/2010/10/25

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Ladder Mix

Manufacturer specifications

Other Knowns

  • well width (24-tooth comb) = 6mm
  • Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye

Calculations

  • 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
  • Per well: 0.6µL ladder + 4.4µL H2O + 1.5µL loading dye = 6.5µL total ladder mix
  • (loading dye = xylene cyanol and bromophenol, made by Bohonak lab)

Make Ladder Mix

  • 120µL ladder + 880µL H2O + 300µL loading dye = 1300µL ladder mix

Gel Scun2 F-R

Cast Gel

  • 270mL TAE + 4.05g agarose in 500mL Erlenmyer flask
  • large stirbar, hotplate set at 150, stir set at 15 for about 45 min, until clear
  • add 27µL GelRed
  • let cool in dark for about 30 min
  • pour into caster, set 2 24-tooth combs, cover with cardboard
  • let cool in dark (had to do office hours, sat for about 2 hours)

Load Gel

Run Gel

  • use top volt-box
  • Power On
    DC On
    Voltmeter: push to right
    Adjust red knob to 90-120V (using red scale of left-side gauge)
  • 90V 3p-4:30p
    120V 4:30p-4:45p

Image Gel

  • nothing...

What Happened?

  • UV light?
    • should be working, when turned off, image black, when turned on, can see gel (but only agarose gel)
  • Gel Stain?
    • used as according to Clark's protocol (10% volume of 3x Gel Red)
    • check product protocol
  • Next:
    • try a bunch of different trials of ladder, gel stain, on small gel




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