How to guide for Laboratory Technicians/Teachers What is Biotech out of the Box? It is a set of equipment -tanks, power packs, pipettors, materials and methods for electrophoresis safely simulating DNA analysis in high school labs What is in each Box? 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 NaHCO 3 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 NaHCO 3 MW (NaHCO3) = 84.01g/L = 1M 0.01M (10mM) = 0.84g/L For 3L = 3 x 0.84g = 2.52g NaHCO 3 2. Dissolve NaHCO 3 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 NaHCO 3 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 NaHCO 3. 2. Heat the agar and NaHCO 3 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 prewarmed 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 NaHCO 3 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.)