Lab 305: DNA Fingerprinting
Part II: Cast an agarose gel
Part II and III, Day 2
Part III: Load an agarose gel
- Prepare your casting tray ("gates" up) and insert a 6-well comb in the end slots.
- Obtain a beaker with 25-30 mL of liquid agarose,
which has been kept at 65-70°C in a water bath. Pour the agarose evenly into the casting tray.
- DO NOT JAR or MOVE the casting tray as the gel solidifies. This ensures a smooth,
even gel. As the agarose polymerizes (about 10 min), it changes from clear to slightly opaque.
- While you are waiting, fill the plastic electrophoresis box with about 300 mL of
1X TAE or TBE --electrophoresis running buffer.
- When the gel has solidified, lower the "gates" on the casting tray and submerge the casting tray onto its platform in the gel box. The comb should be located at the cathode end (black lead; (-) end). The level of the buffer should be only a few mm above the surface of the gel. Add more buffer if needed.
- Carefully remove the comb from the gel (pull it straight out).
You'll notice that this left behind six little empty "slots" or wells in the gel.
- To each of your five tubes, add 2 µL loading dye. Then give them all a quick spin in the microcentrifuge to mix in the dye. (Be sure to BALANCE the tubes in the rotor!)
- Is your gel ready to load? It should be in the gel box, under buffer solution; the comb should have been removed and the six empty wells in the gel should be at the cathode end of the box. You have five samples to load, so you can see that you will have one extra lane. (If you like, use it to practice loading 4 µL LOADING DYE ONLY.)
By convention, DNA gels are read from left to right, with the wells located at the top of the
gel. With your gel lined up in its box with the wells to your left, the contents of Tube "1" should
be loaded in the well closest to you. Thus, when the gel is turned so that the wells are at the top,
"1" will be in the left-hand corner.
- Load 12 µL of each sample into a separate well in the gel.
- Fill your pipet tip correctly to 12 µL.
- Position your arm and pipet over the gel box. Steady that arm with your other arm.
- Lower the pipet tip under the surface of the buffer to the top edge of a well.
BE VERY CAREFUL NOT TO PUNCTURE THE BOTTOM OF THE GEL.
- Gently depress the pipet plunger and slowly expel a sample into a well. Keep the plunger depressed while removing the pipet tip out of the buffer.
- Change tips between samples.