Equipment Glossary

Centrifuge:

aka microcentrifuge - used to seperate reagents

Operation of centrifuge

1. Plug centrifuge into its power supply
2. Plug power supply in to wall outlet. Everything should be on (there is no on/off switch)
3. Load samples and ensure they are properly balanced (equal volumes loaded directly opposite each other). Please supervise students when using the centrifuge to ensure they balance samples properly.
4. Press inner lid on (one of the centrifuges has an inner lid while the other is missing it) and close centrifuge cover.
5. RPM display should read "13". Leave as is for all uses except for cell pelleting during transformation protocol. Use up and down arrows to adjust the speed (rpm).
6. Mode should have RPM lit, the mode lights (rpm and g) are faint on one centrifuge model.
7. Set minutes appropriately.
8. Press Start symbol when ready. It should beep when done and the lid will then unlock.

aka: thermal cycler

PCR machine operation

Plug in and toggle the switch at the back if the machine doesn’t turn on.
Put samples in smaller holes, close lid and lock (switch to tube symbol with round lid)
Once turned on the display should read ‘Main-menu Outreach’
To select the Outreach program hit ‘Enter’, will see a menu with ‘Outreach’ selected
Hit Enter again to start the program, display will say ‘testing program’ before starting to cycle.
If the initial display does not read ‘Main-menu’, press ‘Exit’ until it does and follow instructions as above.

Place gel on transilluminator in plastic bag and place clear shield over it to block the UV rays.
Turn on the transilluminator.

WHEN PACKING UP THE TRANSILLUMINATOR, do not place anything on top of the plexiglass as this has been broken in the past!

Plug in.
Speed can stay at max (7-8) for all uses.

Vortex can be used on "touch" which goes when a sample is pressed into it, or "ON" where it operates continuously.

Preparing your Gel Mold:
There are several styles of gel boxes and accompanying gel molds.

One style of mold needs to be sealed with tape (pressing very well so it does not leak) before pouring the liquid into it. 
Before you begin making the gel, seal the ends of the mold with tape. Some teachers have placed the mold sideways in the gel box, laid saran wrap over the mold and then poured the melted agarose into that. Remember to place the comb in the mold before you pour in the gel and to remove the saran wrap (leaving the gel in the mold) once the gel has set.

The other style (blue mold) has plastic dams that fit into slots to seal the mold so it will hold the liquid agarose and not leak. This mold is placed in the box with the open sides on the left and right (not sideways like the other gel). Slide the two plastic dams into the slots on either end of the mold. Try putting them in at the same time as doing them one at a time may not create a good enough seal. Be sure the apparatus is level so you get a uniform thickness in the gel.

When inserting the comb arrange it with the flat side facing the direction of migration (this will be facing toward the positive red electrode). The comb should not touch the bottom of the mold, although it will be close.

NB: We no longer use high speed gels for this experiment.

Gels are best viewed on the same day you run the experiment. The bands will diffuse if they are left overnight.

Connect the leads to the gel boxes before turning on the power. If you attempt to run the power supply when it is not connected to a gel box with buffer in it, the supply will shut itself off. This is a safety feature of the equipment.

Preparing the Agarose Gel:

1. After readying the mold, take 10 ml of 10X TBE and add 90 ml of tap water, making 1X TBE.
2. Open the packet of agarose and put into the microwaveable container.
3. Pour in 80 ml of the 1X TBE and swirl to mix.
4. Microwave on high for approx. 30 seconds. Swirl again, careful the solution will be hot. Microwave another 30 seconds and keep an eye out as it can boil over. The solution should be clear and just on the point of boiling when all the agarose is dissolved.
5. Add 8ul of Syber Safe DNA gel stain (orange solution provided in microfuge tube) and allow to cool for a couple of minutes, make sure you have placed a comb in the mold, then pour the liquid in.

Note: If the agarose begins to solidify before pouring, it can be reheated to melt it.

6. When gel has solidified and cooled (takes approx. 30 minutes) remove the tape and place the mold into the gel box so the end with the wells (comb) is on the left (near the side containing the positive electrode). In the case of the blue mold, simply remove the plastic dams. The gel could now be wrapped in saran wrap and stored in the fridge overnight until needed.

The gels can be made up ahead of time and it takes about an hour to make two or three.

Simply store your gels covered in plastic wrap in a fridge overnight.

Running and Viewing a Gel

1. Prepare approx. 1 L of 1X TBE (by diluting the 10X TBE concentrate supplied).
2. Verify the gel mold is in the gel box with the wells at the end next to the positive electrode (ie on the left)  and pour in the 1X TBE. Use enough to cover the gel by a few millimeters. Remove the comb.
3. After students load their samples, be sure to prepare the DNA marker by adding loading dye and place it in one of the wells.
4. Connect the leads from the gel box to the power supply (black to black etc). Plug in power supply. Note: Only the model labeled VWR has an ON switch (the large button with horizontal line) the others don’t.
5. For the EC105 model you need to select the ‘low’ voltage range and then use the dial to set voltage. Make sure all selections are set to Volts.
6. The power setting for a Low Speed Gel is 150V.
7. Press RUN to start VWR model, the EC105 will automatically run once connected to gel.
8. When the gel has finished (takes 30 – 40 min.) place it in a sealed Ziploc bag for ease of viewing in the transilluminator. Return the gel with the kit as it is a hazardous waste.

Cleaning:
Rinse the gel apparatus well in running tap water being careful not to damage the platinum wire running along one corner on the inside of the gel box.

Biotechnology in the Classroom is coordinated by the
Department of Biochemistry in the

Schulich School of Medicine & Dentistry at
The University of Western Ontario,
London, Ontario Canada