Objective:
Use techniques such as electrophoresis and restriction mapping to understand the concepts of DNA digestion. Also, become familiarized with restriction enzymes and double digestion.
Results:
The electrophoresis of digested phage lambda DNA is shown in the picture below. DNA has a negative charge and will attract to the positive pole at the end of the gel. The bands in each lane represent different sized DNA fragments. The closest band to the wells represent longer fragments because they can’t travel as fast. The farther away from the well, closer to the positive pole, are smaller fragments of DNA. The farthest lane to the left is the ladder, used to compare different sized fragments. EcoRI has one very distinct band at the beginning
The unknown bacterium that was handed out by the professor labeled “E19” was an irregular and raised shaped bacteria with a smooth texture and it had a white creamy color. The slant growth pattern was filiform and there was a turbid growth in the broth. After all the tests were complete and the results were compared the unknown bacterium was defined as Shigella sonnei. The results that narrowed it down the most were the gram stain, the lactose fermentation test, the citrate utilization test and the indole test. The results for each of the tests performed are listed in Table 1.1 below.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
in the sample in to many identical samples. The DNA retrieved from the reaction can then be
As the solution pH can influence the stability of NaClO-NH3 blend and the elimination of SO2, NOx, the impact of the pH of NaClO-NH3 blend solution on the instantaneous removal as well as the duration time was investigated, and the final pH after reaction was also detected and shown in Fig. 5. It can be seen that the variation of solution pH has a negligible effect on the desulfurization, but the elevated pH has a great promotion on the NOx removal, the efficiencies are significantly increased from 36% to 99% for NO2 in the pH range of 5–12 and from 19% to 65% for NO when the pH is between 5 and 10, after where, both of them are constant. Hence, the optimal pH of the NaClO-NH3 solution for the
Many things have impacted both the Science and Medical fields of study. Electrophoresis and DNA Sequencing are two of these things. Together they have simultaneously impacted both of these fields. On one hand, there is Electrophoresis. Electrophoresis is a specific method of separating molecules by their size through the application of an electric field. It causes molecules to migrate at a rate and distance dependent on their size. On the other hand, there is DNA Sequencing. DNA Sequencing is a technique used to determine the exact sequence of bases
In biology class, we were learning about enzymes. Enzymes are proteins that help catalyze chemical reactions in our bodies. In the lab, we were testing the relationship between the enzyme catalase and the rate of a chemical reaction. We predicted that if there was a higher percentage of enzyme concentration, then the rate of chemical reaction would increase or it would take less time. We placed 1 ml of hydrogen peroxide into four depressions. Underneath the first depression, we place 1 ml of 100% catalase and make 50% dilution with 0.5 ml of water. We take 50% of that solution and dilute with 0.5 ml of water and we repeat it two more times. there were four depressions filled with catalase: 100%, 50%, 25% , 12.5 % with the last three diluted
Gene cloning works by first isolating the desired gene and ‘cutting’ it from the original chromosome using restriction enzymes. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined to the vector DNA by ligation. The vector is introduced into a host cell, often a bacteria or yeast, by a process called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple copies of the inserted DNA. The vector DNA is separated from the host cells’ DNA and purified.
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
Lyons, R.H. (2004). How do we Sequence DNA? In A Primer in DNA Structure and Function. Retrieved from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
Agarose gel electrophoresis separates molecules by to their size, shape, and charge. Biomolecules such as DNA, RNA and proteins, are some examples. Buffered samples such as glycerol and glucose are loaded into a gel. An electrical current is placed across the gel. The current moves the molecules towards the cathode or anode. The speed of the moving molecules depends on the size, shape, and charge. The properties of the gel will definitely affect the movement. Small molecules are expected to move easily and faster thru the pores.
The digestion lab experiment was conducted for the purpose of understanding digestive enzymes and how they work, and under which conditions they function best. Digestive enzymes are present in the body’s gastrointestinal system and mainly function to break down food into nutrients to be absorbed by the body (Oxford Journal, “The Effect of Enzymes on Digestion). The organs that secrete and/or make use of these digestive enzymes are the mouth, esophagus, stomach, small intestine, large intestine, liver, pancreas, and gallbladder. These organs collectively make up the gastrointestinal system, along with the rectum and anus. The functions of these organs will be introduced as the digestive process is explained ahead. The digestive process begins
My hypothesis was I think that if we change the temperature of the enzyme it will have an area where the enzyme works very well, but if we increase or decrease the temperature too much, it will slow the productivity. If we change the pH of the enzyme, there will be an area where it is extremely productive, but if it gets too acidic or basic the productivity will slow. If we change the concentration by increasing the amount of enzymes, it will be more productive. T think this because your are having more than 1 enzyme work on one task. I you decrease the amount of enzymes, the productivity will be slower because there are less enzymes working on that certain task. The data refutes my hypothesis because I found that the enzymes work best in one
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.