We are going to compare the fragments of viral DNA by adding three different enzymes.

solution into an enzyme tube of your choice (refer to Fig. 4 for the colour code). Mix the liquid and the dried enzyme by carefully drawing the liquid up and down in the tip a few times.Repeat this for each enzyme tube and the yellow ‘control’ tube, using a fresh tip each time to prevent cross-contamination between the tubes.Cap each tube with an appropriately coloured lid.Incubate the tubes at 37 C, in a water bath or incubator, for between 30 and 45 minutes.Place the electrophoresis tank on a level surface, where you can leave it undisturbed for the next 20–30 minutes.Slot a 4-toothed comb in place at one end of the tank.Pour about 10 cm3 of molten agarose into the tank so that it fills the central cavity and flows under and between the teeth of the comb. Try not to spill liquid into the areas at either end.Leave the tank undisturbed until the gel has set hard.Get tow electrodes for the electrophoresis tank.Pour slightly more than 10 cm3 of TBE buffer solution into the electrophoresis tank. The liquid should just cover the surface of the gel and flood into the areas that will hold the electrodes.Very gently ease the comb from the gel, allowing the buffer solution to fill the wells left behind. Take care not to tear the wells.Put the tank where you are going to ‘run’ it, where it will remain undisturbed.Put a clean tip on the microsyringe. Add 2 ul of loading dye to the tube containing the DNA you wish to load. Mix the dye and the DNA sample thoroughly, by drawing the mixture up and down in the microsyringe tip.Pipette the loading dye/DNA mixture into one of the wells, holding the tip above the well but under the buffer solution.Make a note of which DNA you have put into the well.Repeat the last two steps with each DNA digest and the ‘control’ tube with no restriction enzyme. Remember to use a new tip for each digest, to avoid cross-contamination.Fit one electrode at each end of the tank as shown in the illustration. Join the electrodes to batteries using wires with crocodile clips. Sufficient batteries should be used to give 18 volts.Check that contact is made between the buffer solution and the electrodes. Leave the gel to ‘run’, undisturbed, for seven hours.Disconnect the batteries once the blue loading dye has reached the end of the gel. Rinse the crocodile clips in tap water and dry them thoroughly to prevent corrosion.Pour about 10 cm3 of staining solution onto the surface of the gel.Leave it for exactly 4 minutes.Wash surplus stain from the gel surface with about 5 cm3 of 70% ethanol for a few seconds Pour away the alcohol, and then rinse the gel very carefully with cold tap water. Rinse with water 3 or 4 times to remove any excess stain.The remaining stain will gradually move down through the gel, staining the DNA as it does so. Faint bands should start to appear after 10 minutes. The best results will be seen if the gel is left to ‘develop’ overnight. (Put the tank in a plastic bag if you do this, to prevent the gel from drying out.).                  Fig. 2 (Water bath) Fig. 1 (Microsyringe)      Fig. 4 (Colour code for the tubes) Fig. 3 (electrophoresis tank) Data recorded: Enzyme usedEcoRIBamHIHind IIIControlLength of movement along the tank 10.8 cm30.9 cm30.7 cm30.6 cm3Length of movement along the tank 21.0 cm31.7 cm31.3 cm3Length of movement along the tank 31.4 cm32.0 cm31.6 cm3Length of movement along the tank 41.5 cm32.2 cm3Length of movement along the tank 51.8 cm3Number of base pair fragments (bp)6681                                                                                       Photographs of the gel with the stains Sketch of the gel with the stains Graph of base pairs against the migrated distance of the fragments of DNA Conclusion: The fragments that moved more across the gel were the ones that had less base pairs because their mass is smaller and they can move across the gel more easily than the fragments that had more base pairs. So the first fragment in the gel is going to be the one with the most base pairs followed by the one with the second biggest number of base pairs. The control was the one that moved less because it had the complete strand of the Lambda DNA and so is the one that had the most base pairs and it had more problem to move along the gel than the other stains that are only fragments of the Lambda DNA. Some of the fragments were “missing” because they were not stain with the dye or they are too small to be seen by the human eye. With the graph that was made using the data the average data of Hind III we can know how much would any fragment of the Lambda DNA of any size would move across the gel Problems with the experiment: Some of the Lambda DNA was not stain or they were too small to be seen, this problem could be resolved by using a fluorescent gel that can stain more all the Lambda DNA fragments. Also some of the lines in the gel were too close to each other and it was had to identify them, this could be resolved by using a gel with a lower density than the one that we used. Restriction map of the enzymes used Average of the distance travelled by the fragments of Hind IIIFragment 10.68 cmFragment 21.03 cmFragment 31.42 cmFragment 41.63 cmFragment 52.00 cmFragment 62.60 cm This is the average for the howl class