Usage Of Restriction Enzymes Of Dna Fingerprinting

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

The idea behind Gel Electrophoresis is that we inject a slab of gel with the DNA we found at the crime scene. We then inject the same gel, next to the crime scene DNA, with suspect 1’s DNA and suspect 2’s DNA. We then send an electric current through the gel and wait for the results. The smaller molecules in the DNA will travel farther than the bigger molecules because the bigger ones will have difficulty making its way through the microscopic beads in the gel. After the separate bands appear in the gel, we stain it with a special chemical called Ethidium Bromide to give it a color under the blue

The purpose of this lab is to test for enzyme activity by examining factors that may influence enzymes.

There were a couple of problems that our group encountered while conducting this experiment. Since this was our first experiment dealing with enzyme activity, the probability of human error increased. We found it difficult to go through the procedures with undefined roles for our team. The other problem that our team encountered was the getting the cuvettes into the spectrophotometer quickly once the enzyme was introduced to the cuvette.

To tube 2 and tube 3 a boiling chip is added. The two tubes are boiled to remove any residual ether. Next, the tubes are cooled to room temperature and placed into an ice bath to allow for crystallization. The solution is then removed from the solid in each tube and discarded. To tube 2 and 3 ~0.5 ml of H2O is added for recrystallization, the tubes

We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the

Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.

These steps will repeat for every enzyme concentration’s control, substrate and enzyme test tubes. The total amount of data points should be 6, 2 data points per concentration. After part one, clean the test tubes by correctly disposing of the substances in the waste bin under the fume hood and then proceed to clean with soap so that the test tubes can be used for part

After recognizing the restriction sites, the restriction enzymes cut off the DNA strands at these points, leaving sticky ends on the original DNA fragment. The restriction enzymes can also travel to other cells to cut off DNA sequences, the sequences that can match the bases of the original sequence. The sticky end will find other sticky end to form a new recombined DNA sequence, obeying the law of base-pairing.

The first agarose gel provided insight into whether or not Zassy’s genomic DNA had been cut by the Sal I HF (NEB) restriction enzyme. As seen in Figure 1, Zassy’s cut DNA, in lane 3 left clear fragments between the 3 kb and 0.5 kb areas. Zassy’s uncut DNA, in lane 2, left a clear and bright band above the 10 kb marker. Therefore, the gel confirmed that Zassy’s DNA was successfully cut with Sal I because there was a significant difference the cut and uncut DNA that was run through the gel. There was an additional lane of cut DNA because another student adopted Zassy in the early stages of this experiment. Nevertheless, the clear distinction between lanes 2 and 3 allowed the experiment to continue on to purification of the DNA.

Following this action, in tubes 1 and 3 15-µl of Enzyme 1 was dispensed and the same amount was dispensed for tubes 2 and 4 of Enzyme 2. The final volumes of the each of the reaction tubes had a final volume of 40-µl (Table 1). After dispensing what was needed in each of the tubes, one of the team members tapped each tube to mix all the contents together and to be sure that the contents were at the bottom of each tube. Sequentially, after all the contents were mixed and were at the bottom, the tubes were then placed in an incubator at 37°C for 45 minutes (Upadhayaya, 2016). Prior to removing the DNA samples from the incubator, the Gel for Electrophoresis had to be casted.

Once each test tube had been in the respective temperatures for a minimum of 10 minutes, test tube 1 was taken out of its bath and the rest of the tubes were left in their respective places. To find the rate of enzyme activity for test tube 1, 10 drops of enzyme solution were added, and the timer began. The contents were poured into a clean 250mL Naglene bottle, and swirled to mix. Next, the O2 Gas Sensor

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

Recent advancements in DNA technology have improved law enforcement’s ability to use DNA to solve old cases. Original forensic applications of DNA analysis were developed using a technology called restriction fragment length polymorphism (RFLP). Although very old cases (more than 10 years) may not have had RFLP analysis done, this kind of DNA testing may have been attempted on more recent unsolved cases. However, because RFLP analysis required a relatively large quantity of DNA, testing may not have been successful. Similarly, biological evidence deemed insufficient in size for testing may not have been previously submitted for testing. Also, if a biological sample was degraded by environmental factors such as dirt or mold, RFLP analysis may have been unsuccessful at yielding a result (Turman).

DNA fingerprinting is a scientific technology involving the extraction, replication and arrangement of strands of an organism’s DNA. This results in the formation of a genetically distinctive fingerprint that is unique to the organism which the DNA sample was originally extracted from. Because of the specificity of a DNA fingerprint, the application of this technology can have a substantial influence on many aspects of society. Accessibility to a DNA database allows for higher efficiency in forensic investigations, personal identification, maternal and paternal testing. The availability of a national database to police officers and forensic scientists would equate to increased productivity in investigations and prosecution of suspects in a