Dhruv Chopra

U68 Core Practical: Gel electrophoresis

Biology

Aim: To show the effects of different restriction enzymes on separate samples of lambda DNA through gel electrophoresis. Apparatus required: Microsyringe 8 sterilised graduated microsyringe tips Electrophoresis tank 4 restriction enzyme tubes Foam floater Water bath set at approx 37oC Carbon fibre tissue Electrical Power Pack (wires + electrodes included)

Method 1. Add 100 l of distilled water to the lambda DNA tube using the 10 l graduation on the microsyringe. 2. Leave the DNA tube upright for 5 minutes. 3. Flick the bottom of the tube for 1 minute whilst holding the tube firmly to mix the distilled water and DNA. 4. Leave the DNA tube for another 5 minutes. The DNA and distilled water must have formed an opaque solution. If not, then use the microsyringe to draw the mixture up and down the tube a couple of times. 5. Put a new graduated tip on the microsyringe and add 20 l of lambda DNA mixture to each of the four enzyme tubes (EcoRI, BamHI, HindIII and a control tube) and mix the liquid by drawing it up and down the microsyringe. With each enzyme tube use a fresh tip to reduce the chances of contamination. 6. Melt agarose gel and put it in a sealed container water bath at 55 to 60 oC 7. Put the electrophoresis gel tank on a level surface and pour the molten agarose gel inside slowly with the comb slotted at the far side of the tank to create the grooves, just enough to fill the central area. 8. Leave the tank until the liquid has set and turned milky. Cut two pieces of carbon fibre of 42mm x 22mm each. These will be the electrodes.

Core Practical Write Up

2011

Dhruv Chopra

U68

Biology

9. Add 10ml of TBE buffer solution to the gel tank after the agarose gel has set. Gently remove the comb before doing this to allow the solution to fill the grooves. 10.Placing the tank under a piece of black card for better visibility, use the microsyringe to add 2 l of loading dye into each of the enzyme tubes, taking care to use a fresh tip for each and mixing the two thoroughly. 11.Pipette each mixture carefully into a specific groove, noting down which enzyme corresponds to which groove, taking care not to spill the mixture in the gel. 12.Connect electrodes to the edges of the tank making sure they connect to the carbon fibre at the sides. 13.Use a 36 volt transformer on the power pack and make sure that the DNA is on the cathode so it will be attracted to the anode and move across due to the electric gradient. 14.After 2 hours, switch off the power and remove the electrodes. Put on plastic gloves to prevent the stain from touching the skin for safety. 15.Pour 10ml of staining solution (0.4% Azure A in 20% ethanol) onto the surface of the gel and leave for 4 minutes. 16.When the 4 minutes are exactly up, put the stain in a beaker to be returned to the bottle for re-use. 17.Wash the surplus stain away with approx 5ml of 70% ethanol, then rinse this away with distilled water about 3 or 4 times, taking care to leave as little water as possible on the surface of the gel as this will remove the stain from the top of the gel. 18.Put the tank in a plastic bag to prevent the stain from drying out and place in a dark, cool place.

Core Practical Write Up

2011

Dhruv Chopra Results and interpretation of results

U68

Biology

We were not able to find any clear fragments of DNA visible in the tank but there was some stain that had travelled towards the anode on the control and EcoRI tests. This may have been a result of several factors that contributed to this: cross contamination whilst mixing the enzymes with distilled water or when adding the dye to the enzyme mixture, poor electrical equipment and use of electrical equipment or human error whilst pipetting the mixture into the grooves of the gel tank (damaging the grooves or spilling dye). Overall the aim of the experiment failed as there were no visible fragments.

Core Practical Write Up

2011