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

{| width="800"
 * style="background-color: #BCED91"|[[Image:owwnotebook_icon.png|128px]] Tara's Lab Notebook
 * style="background-color: #9DB68C" align="center"|  |Main project page
 * style="background-color: #9DB68C" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Manufacturer specifications

 * 0.1µg ladder / mm width of well
 * STK at 1.0µg/µL
 * [[Media:Invitrogen_1KbLadderProductManual.pdf]]

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

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

 * [[Image:BlankGelSchematicTop.jpg|600 px]]

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...
 * [[Image:20101025_Top.TIF|300 px]] [[Image:20101025_Bottom.TIF|300 px]]

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


 * }