Restriction digestion of DNA and agarose gel electrophoresis
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Published: Thu, 04 May 2017
In this experiment, EcoR1 which is the restriction enzyme is used. The function of restriction enzyme is to cut the DNA into smaller stand. Hence, in this experiment, DNA pBR322 is been cut by the restriction enzyme into smaller DNA strand. This can be seen from the picture above which the fluorescent colour of the uncut plasmid is brighter compared to the cut plasmid. The uncut plasmid contain brighter colour is due to the concentration and the thickness of DNA which coiled together. From the original plasmid, DNA pBR322 which the DNA stand is in circular strand, it is now been cut by restriction enzyme and form single band of plasmid, DNA pBR322 and the fluorescent colour is less bright.
As the result of this experiment, there is only one band is produce after adding the restriction enzyme, EcoR1. Originally, the DNA plasmid’s structure is in singular circle. Then, the restriction enzyme is added which to cut the specific site which is 4359bp of the pBR322. Hence, this lead to forming of one linear band. Hence, there is different in number of band between cut and uncut. While for the band size, the uncut DNA plasmid seems to have a brighter fluorescent colour when show under the UV light. While the cut DNA plasmid shows less bright. The brightness of the fluorescent colour can determine the thickness and the concentration of the DNA plasmid. Therefore, the uncut DNA plasmid will have more concentration and thinker compared to the cut DNA plasmid.
Besides, there also have a possibility of having an experimental error. The experimental error is there might have adding different type of restriction enzyme other than EcoR1. There might be accidentally adding Eco321 which this restriction enzyme cutting different part of the plasmid and causes the plasmid to produce two band. Therefore, this leads to have an error result which produces two bands instead of producing one band.
Electrophoresis enables the DNA to move through gel matrix. Hence, during agarose gel electrophoresis, the DNA is place at the cathode. This is because the DNA will be pulled toward the positive terminal due to the DNA consist of phosphate group which is negatively charge and that’s the reason of DNA being attracted towards the anode of the electrophoresis chamber. While pulling, the electromotive force produce by the current is help to speed up the DNA migration to cathode.
Moreover, smaller fragment of DNA move faster towards the anode compared to the larger fragment of DNA. The agarose gel is a solid material. Therefore, the smaller fragment of DNA able to squeeze through the gel easily compared to the longer fragment of DNA. Besides, by observing the speed of movement of the DNA towards cathode, this can also determine the band size of the DNA molecule.
Q2. What is a restriction enzyme? What structure does it recognize? What chemical bond does it cleave? Name one other example of restriction enzyme name that gives sticky ends. (4m)
Restriction enzyme is act as a scissors which cut the DNA. It recognize a specific region of DNA. The chemical bond it cleave is by hydrogen bond. Another example of restriction enzyme name that gives sticky end isPst 1.
Q3. What is the use of gel electrophoresis buffer? (2m)
The use of gel electrophoresis buffer is to provide ion which enable the current to pass through and complete the circuit.While some other type of buffer is to maintain the pH in the solution which suitable for the enzyme.
Q4. Why is it important to include a reaction with no added restriction enzyme (an ‘uncut’ control)?
So that enableto compared the different between the added restriction enzyme and the no added restriction enzyme.
Q5. What is the use of the loading dye? (2m)
The loading dye is less dense compared to the DNA. Hence, the DNA will sink in the well. Besides, loading dye enable to tell when is time to switch off the electrophoresis chamber.
Q6. How are the DNA bands viewed? What is the use of SYBR® Safe DNA gel stain? How does it work? How different is it from loading dye? (5m)
The DNA bands is viewed by blue light transilluminator or standard UV translluminator with the glass on top to prevent being harmful to the eyes. The use of SYBR® Safe DNA gel stain is similar as a loading dye in fact that they are environmental safe due to not state as hazardous waste. SYBR® Safe DNA gel stain different from the loading dye is because SYBR® Safe DNA gel stain able to stain the DNA while the loading dye is only can give the colour of the solution but not the DNA.
Q7. How is SYBR® Safe DNA gel stain better than the conventional ethidium bromide staining method?
SYBR® Safe DNA gel stain better than the conventional ethidium bromide staining method because SYBR® Safe DNA gel stain is make to reduce the mutagenicity compared to ethidium bromide which is highly carcinogenic and a strong mutagen to the DNA. Besides, the detection sensitivity of SYBR® Safe DNA gel stain can compared to ethidium bromide. Other than that, SYBR® Safe DNA gel stain is more safer compared to ethidium bromide because SYBR® Safe DNA gel stain have no acute toxicity.
Q8. State the advantages of using bacterial DNA in recombinant DNA technology. (5m)
Bacterial DNA can be produce faster compared to others due to the circular strand of DNA. Other than that, the DNA can be easily cut by the restriction enzyme according to the restriction map. Besides, bacterial DNA can be used to produce more food such as hormone bovine samatotropin is injected into the cow by bacterial DNA so that the cow will produce more milk.
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