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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