When using the microcentrifuge:- Do not open the centrifuge until it stops completely.
- If the centrifuge tubes are smaller than the metal
holder or holes, use the proper adaptor to
accommodate them. - Do not unplug the centrifuge by pulling on the
cord. Pull the plug.
- Place the tubes in a 37 °C water bath for a minimum
of 45 min. Use a thermometer to check the
temperature of the water. - Once the digestion is complete, place the tubes in
the polystyrene cup and put the cup in a freezer until
your next class. Make sure you have labelled your cup
with your name.
Day 2: Gel Electrophoresis
- Measure 0.96 g of agarose powder in a paper boat on
an electronic balance and transfer to a 500 mL
Erlenmeyer flask. - Use a graduated cylinder to add 125 mL of 1TBE
buffer and swirl to mix. - Heat the flask on a hot plate or in a microwave until
the solution is completely clear. Handle carefully,
using tongs or oven mitts. Make sure you wear
goggles and a lab coat.
If the agarose gets too hot it may bubble over. Be
sure to observe your Erlenmeyer flask throughout
the heating process. If the agarose solution starts
to bubble up the neck of the flask, remove it
immediately from the heat source using an oven
mitt or tongs. Handle all hot glassware with caution.- Prepare the gel casting tray. Depending on your gel
electrophoresis unit, you may have to tape the gel
casting tray. Ensure that the plastic comb is inserted
properly. - Once the flask with agarose solution is cool enough
to handle with bare hands, pour the mixture into the
gel casting tray. The comb teeth should be immersed
in about 6 mm of agarose. The gel should cover only
about one-third of the height of the comb teeth. Use
a micropipette tip to remove bubbles from the gel as
soon as it is poured.
INVESTIGATION 20.2continued 12. Allow the agarose to set for a minimum of 20 min.
The gel will become cloudy as it solidifies.- Once the gel has set (you may test this by gently
touching the lower righthand corner with your
finger), flood the gel with 1TBE running buffer
and then pull out the comb gently without ripping
any of the wells. - Orient the tray containing the gel in the gel
electrophoresis box so that the wells made by the
comb are at the end with the positive electrode. - Add 1TBE buffer to the gel electrophoresis box
until the buffer is approximately 5 mm above the gel.
Place the gel electrophoresis box to the side. - Add 1 L of loading dye to each of the Eppendorf
tubes. Microfuge for 3 s. - Micropipette the full contents of one Eppendorf tube
into a well on the gel. Do the same for each tube. Be
sure to record the order in which you dispense the
tubes. Steady the micropipette over each well using
both hands. - Close the gel box and connect it to the power supply.
If you are using a gel box that you made, set the
voltage to 45 V dc and turn it on. Electrophorese
for 12 h. Alternatively, if you have a stronger power
supply or a store-bought electrophoresis unit,
electrophorese at 110 V for 2.5 h.
When using the power supply:- Be sure the grounding pin in the power supply is
not broken. - Pull the plug, not the cord, when unplugging the
power source. - Do not let the wire leads connected to the electric
power supply or batteries touch each other.
- Unplug the power supply and carefully remove the
gel. Wrap the gel in plastic wrap and place it in the
refrigerator for a maximum of one day.
Day 3: Staining the Gel
- Unwrap the gel and place it in the staining tray.
- Flood the gel with 0.025 % methylene blue solution.
Let the gel sit in the solution for at least 20 to
25 min. Pour off the water and replace it with fresh
water. Repeat this process three more times. Keep an
eye on the intensity of the DNA bands. If you destain
for too long, you may lose the smaller fragments.
698 Chapter 20 NEL