Gel Visualiation¶
| Author: | Carl Oliveros |
|---|---|
| Copyright: | This documentation is available under a Creative Commons (CC-BY) license. |
Modification History¶
Purpose¶
To visualize either DNA extracts, PCR amplicons, or DNA libraries for quality assurance.
Steps¶
Prepare 1.25% agarose solution in an Erlenmeyer flask, by mixing the following:
Small gel (in 50 mL flask) Big gel (in 250 mL flask) 0.5 g agarose powder 2.0 g agarose powder 40 mL 1X TBE buffer 160 mL 1X TBE buffer Add GelRed (1 μL for small gel, 2 μL for big gel) to the solution in each flask. Mix by swirling.
Heat up solution in a microwave until solution is homogenous (~ 1 min for small gel, ~ 2.5 min for big gel). Mix by swirling. This should take several (2-3) cycles of microwaving & swirling, microwaving & swirling to homogenize.
Cool gel to ~ 60°C using a waterbath, running cold water over the flask, or by sitting on the counter and letting it cool until you can touch the glass without it being extremely warm.
While the gel is cooling, set up the gel bed for casting. Make sure the gel bed is level and that the comb is seated correctly.
Once gel in flask has cooled to a reasonable temperature, pour warm gel on to gel bed and wait until gel solidifies (15–20 minutes).
While gel is solidifying, prepare your samples for loading by mixing each DNA sample with 1 μL loading dye.
Remove the comb/s from the gel and transfer the gel (on the gel bed) to the gel rig. Orient the gel so that DNA will run through the gel in the correct direction (away from negative [black] terminal and toward positive [red] terminal). If necessary, add 1X TBE buffer to the gel rig so that the gel is completely immersed in buffer.
Load DNA samples and ladder into the wells of the gel.
Place the lid of the gel rig securely. Connect the terminals to the power box. Run the gel at 110–120 V for ~1 hour.
Take a photo of the gel using the imager in the 3rd floor common equipment lab.