“How to” guide for Laboratory Technicians/Teachers
What is Biotech
out of the Box?
What is in
each Box?
It is a set of equipment -tanks, power
packs, pipettors, materials and methods
for electrophoresis – safely simulating
DNA analysis in high school labs
1 class-room set (3-4 students/group)
2 power packs
8 gel tanks
8 pipettors and boxes of tips
30g agar powder to make gels
8 tube racks and bag of sample tubes
2ml stock solutions of dyes
1 bag transfer pipettes
1ml stock solution of blue dye for
preparing blue water for pipetting
practice
1. Prepare bicarbonate buffer
10mM NaHCO3 Solution - Bicarbonate buffer is used to make the agar gels and to fill the tanks.
For one class making 8 gels (8 x 35mL) and filling 8 gel tanks(8 x 300mL) you need to make 3L
buffer.
1. Weigh 2.52g NaHCO3
MW (NaHCO3) = 84.01g/L = 1M
0.01M (10mM) = 0.84g/L
For 3L = 3 x 0.84g = 2.52g NaHCO3
2. Dissolve NaHCO3 in distilled water and make
up to 3L
3. Label and store at 4°C
2. Pipette samples and put into racks for students
The dye solutions are supplied to laboratory
technicians as stock solutions in screw-cap vials. Each
stock solution is comprised of a coloured dye dissolved
in 20% glycerol so the samples sink into the sample
wells.
There is one ladder, made by combining all 5 dyes (this
has been done for you) and 5 stock dye solutions from
which several combinations can be made, for example;
• Pair 1; Orange G & Tartrazine
• Pair 2; Bromophenol blue & Rose Bengal
• Pair 3; Rose Bengal & Orange G
A minimum of 100µl of each dye or pair is required for
a class of 8 groups. This allows 10 µl of each dye or
pair per group with some spare.
Aliquot samples for each group of students into the
sample tubes (supplied) and label as suits teacher.
Aliquot slightly extra sample to allow for pipetting
errors for example, aliquot 12ul into the sample tubes
for students to load 10ul into gel.
Labelling for student samples can suit the teaching
plan:
e.g. DNA size ladder (ladder), Whale DNA (Pair 1),
Tuna DNA (Pair 2), unknown Fish DNA 1 (Pair 1),
unknown Fish DNA 2 (Pair 3), unknown fish DNA 3
(Pair 2).
*
The dyes used (at 0.2% w/v) are Bromophenol Blue, Orange G, Rose Bengal, Tartrazine and
Indigo Carmine. Their MSDS safety profile has been checked and they are safe for students.
3.
Make 1% Agar Gels
Reagents


10mM NaHCO3 solution
Bacterial Grade A agar (substitutes for molecular grade agarose to reduce cost) - this is
supplied in each kit.
Method
1. To make eight 1% agar gels, weigh 2.8g
(8 x 0.35g) of bacterial grade agar into a
1L conical flask and add 280mL (8 x
35mL) of 10mM NaHCO3.
2. Heat the agar and NaHCO3 solution in a
microwave oven on medium-low power for
1 minute at a time, swirling between
heating until the agar is dissolved.
Granular/cloudy appearance becomes
clearer and more homogenous as agar
dissolves. Use insulated gloves to hold
the conical flask containing the molten
agar.
3. Prepare the gel-casting trays by placing
them in the gel tanks with the rubber
gaskets sealed at the sides of the gel
tank.
4. Put the sample combs into the slot
closest to the end of the gel tray.
5. Measure 35mL of molten agar into a pre-
warmed measuring cylinder and pour into
each gel tray. Repeat for all 8 tanks
before the solution cools. (Agar can be
rewarmed if needed) “Chase” away any
bubbles with a yellow tip.
6. Allow gels to set completely for 30 mins
at room temperature.
7. Remove gel tray containing gel from tank.
Turn gel tray 90° clockwise, so that the
sample comb is placed over the red
stripe, (at the left-hand side of the
tank.)
Gels can be stored overnight or for a few
days in an air-tight container in the
fridge with a little buffer covering them.
Note: Each student group will need a bottle
of bicarbonate buffer (300mL) to fill their
electrophoresis tank
4. Lay out of student benches
Each power pack will run 4 tanks
5. Electrophoresis of samples (students do this)
Materials
1% agar gel
Bicarbonate buffer
Tube rack containing samples
Micropipette
Box of yellow tips
Discard container for tips
Electrophoresis tank
Electrophoresis power pack (1 per 4 tanks)
Digital camera (optional)
Method
1. Check the agar gel is positioned in the
electrophoresis tank the right way,
(sample comb at left hand side.)
2. Pour in NaHCO3 solution into both
ends of the gel tank to cover the gel
to a depth of about 1 mm.
3. Carefully remove the sample comb without
damaging the wells
4. Using a P20 pipette load 10µL of the ladder
in the first lane/well and 10µL of each
sample into the next wells. The sample will
sink to the bottom of the well as it contains
20% glycerol to make it dense.
5. Place lid on the gel tank and plug the other
ends of the leads into the power pack. The
dyes migrate from the negative cathode
(black lead) towards the positive anode (red
lead.)
6. Set the power pack to run at 90V for 30
minutes. Press the ‘Start/Pause’ button to
start the electrophoresis.
7. After 30 minutes turn off the power and lift
the lid off the gel tank by depressing the
white knobs using your thumbs and lifting
the lid at the same time.
Remove the gel tray from the tank and
interpret the separation patterns. The dyes will
diffuse quickly so either take a photo or freeze
the gel until next lab.
(Gels can be discarded in a rubbish bin.)