Red – EcoRI enzyme
Blue BamHI enzyme
Green – HindIII enzyme
Yellow – no enzyme
- Mix the DNA with the enzyme very precisely ensuring that it got mixed. The best way is to draw the solution up and down in the tip a few times. Do the same thing with all the colored tubes.
- Place an appropriate color lid on top of each tube, stick them into a foam block and put into an incubator at 37˚C for 30 minutes.
- Then we froze the contents of the colored tubes in order to be able to continue the experiment on another lesson.
- Preparing the gel: (Diagram #1)
- At first we placed the electrophoresis tank on a level surface, so that the DNA won’t be moved by a mistake.
- Then we poured over 10 ml of gel into the tank, so that only the spaces at the ends of the tank were not filled with it.
- We allow the gel to set and wait till it’s ready for the next process.
- Loading the Gel: (Diagram #2)
- When the gel is already hard we pour 10 ml of buffer into the tank so that it covers the gel and floods the space at the ends of the tank.
- Then we carefully pour the comb out of the tank and 4 wells are produced, which soon the buffer solution falls into.
- Place a black card under the tank, in order to be able to observe easier.
- We then added 2μ of dye to the DNA in each of the colored tubes and one by one (always exchanging tips when in touch with another liquid) ensure to mix the DNA with the dye by drawing the mixture up and down in the microsyringe tip.
- Very carefully we pipetted each of the mixture to the wells recording, which one was placed in which well. We had to hold the tip below the buffer solution but above the well and attempt to pour a similar amount of solution in each of them.
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Running the Gel: (Diagram #3)
- We cut two carbon fibre electrodes of the same dimensions (22mm/42mm) and fit them at the ends of the tank.
- We attached them to the wall of the tank and 2 9-volt batteries using the wires.
- We ensure that we have connected the positive (+) terminal of the battery to the electode fibre furthest from the wells.
- Check that contact is made between the buffer and the electrodes; add a drop more solution if necessary.
- We left it run for around 12 hours.
Diagram #1 Diagram #2 Diagram #3
- Staining the DNA:
- When the blue dye has reached the end of the gel, take out the electrodes and pour off the buffer solution.
- In order to stain pour 10ml of staining solution onto the gel and leave it for 4 minutes.
- Return dye to bottle and wash of f with 5ml of 70% ethanol.
- Pour off the alcohol and rinse with cold water 3-4 times.
- Draw a diagram of your observations – the movement of the fragments of DNA.
DATA COLLECTION:
Restriction of lambda DNA fragments after electrophoresis
We observe a various movement of the DNA fragments and formulation of DNA bands.
DATA ANALYSIS:
Some other results of DNA fragments separation by electrophoresis showing clearly the various bands.
This are results of electrophoresis of some other DNA.
The white bands represent DNA of a particular size.
Lambda is a virus, it is able to express some of its genes by taking over the bacterial cell that it infects. In this investigation, I was observing the effects of three restriction enzymes on Lambda DNA, such as EcoRI, BamHI and HindIII. After adding the DNA mixture with distilled water to a tube with one of these restriction enzymes, the enzyme searches for a specific sequence of base pairs and cuts it at a specific arrangement of base pairs. This specific DNA arrangement may appear many times and due to that we will be provided the fragments that we are able to separate by electrophoresis.
When different restriction enzymes are used to cut the Lambda DNA, fragments of varying sizes are produced, what we can clearly observe after putting the fragments into gel electrophoresis. DNA is negatively charged due to its phosphate backbone and that’s why the DNA fragments will move toward the positive electrode and create a pattern of DNA bands. The distance these bands move through the gel is determined by its size and weight. Due to what, a small fragment will be able to move quickly, whereas a large fragment will move more slowly. In our forth well we placed the uncut DNA, which we observe didn’t move much, moved the least and didn’t separate. We stained the gel with a chemical that will combine with the DNA causing it to take on a blue color in order to see the pieces easier.
We observe that the movement of the molecule placed in well 4 – the uncut DNA, in the positive charge direction is the shortest when comparing to any of the others. The rest of the Lambda’s DNA cut by specific enzymes created bands, but the separation we observed was minimal and smearing pattern was formed. Many of the groups from our class also didn’t see any movement at all, which was probably caused by no DNA present (explanation in EVALUATION section).
Both in our experiment results and results of some other group we observe bands, which represent the fragments constructed after the separation of the Lambda DNA separated. The higher on the diagrams we look – meaning the closer to the wells and the negative power there are the fragments, which are bigger. E.g. when we look on the picture of the electrophoresis results of some other experiment we sea that the closer from the wells the fragments are the whiter and more vivid they appear on the photo, which refers to its weight and size. The lower we look at on the picture or our results the thinner the fragments are.
To help me understand the results I imagined myself being a DNA molecule. I imagined I was placed in an agarose gel, which is an obstacle on the DNA’s way, almost like a very dense spider web. If I were a small fragment, I could easily crawl through the spaces in between the webs, but if I would be bigger it would be way harder to fit into the spaces. We can conclude that HindIII cuts the DNA in the highest number of places and so it is easier for it to crawl through the aragose gel, while it is almost impossible for the uncut DNA to move.
Electrophoresis, which separated the fragments of the DNA is very important in life and might even save a person’s life. Let’s look on the idea of fingerprinting.
“How is DNA evidence prepared and analyzed in a crime case? Students perform agarose gel electrophoresis to analyze DNA samples from a mock crime scene. Based on DNA fingerprinting profiles that simulate samples (the DNA source is actually a bacterial virus) from the two suspects, the victim, and the blood on the murder weapon, students determine which suspect likely committed the crime. This activity helps students understand how DNA variation in individuals can be analyzed in practical applications such as genetic testing and forensics.”
This is taken from a teacher guide for a lab, which is just a ‘game’, but in reality fingerprinting is used in those ways.
EVALUATION:
We can observe that our results show the main idea of electrophoresis and the pattern can be observed of the smaller pieces moving further in the direction of the positive charge. Although, our results are not perfect and have a lot of weaknesses and possibilities to be modified, I can conclude that my hypothesis was supported. The pattern I expected was shown on results.
Weaknesses and Modifications:
- We might have used a better way to mix the DNA at the beginning with destilled water, because it is very likely to have occurred that the groups, which haven’t observed any DNA movement after electrophoresis, didn’t even achieve to take the DNA out of those tubes. We should have used e.g. a vibrating mixer for scientists.
- The microsyringes we used were rather accurate, but it would have been even more precise and used automatic, controlled with a computer pipettes.
- There is also a possibility that there was no electrical contact.
- We could have used a 100μm pipette instead of ten times using a 10μm pipette.
- We could have left the DNA and restriction enzyme in the incubator for a longer time or maybe just make the experiment using various times.
- We could have also increased the gel volume, what would make the experiment more accurate.
- We should have experimented also with various concentrations of agarose, what we determine the easiness of the DNA fragments passing through the gel.
- We could have used batteries with higher voltage, so that the process would happen faster or we could have observed its influence on electrophoresis.
- Most importantly we should have precisely calculated the distanced the fragments moved and their sizes, but to do that we would need better equipment.
Sources:
I used my own knowledge, my biology book “Advanced Biology for You” by Gareth Williams and such internet sites as:
