Cell lysis solution was used to lyse the cellular membrane. At this stage, the nuclei are still intact. The next step was addition of Puregene Proteinase K after centrifuging .This was added to aid in the digestion of contaminating proteins such nucleases which degrade DNA and the nuclei wall so that we can obtain the DNA from the nucleus and also release the strands of DNA from the histones (6). Protein precipitation solution was then added to precipitate all the cellular debris to obtain only the DNA (7). After another centrifugation step, the left over pellet is the debris while the supernatant contains the DNA. Next, the supernatant was added to the isopropanol and glycogen solution. Isopropanol was added to precipitate the DNA since it is less soluble in isopropanol. However, whatever salts are present in solution have a greater tendency to precipitate as well (8).Glycogen solution is added to maximise the amount of DNA recovered after precipitation (9).Finally, the pellet which is obtained after another round of centrifuging was washed with ethanol which was to remove all traces of glycogen solution as it can affect the purity of the DNA being recovered. DNA hydration is used to hydrate and since pH changes can easily occur, to stabilize the pH A summary of all the solutions and their respective functions are shown
Our materials for the extraction of DNA included: the screwcap tubes, capless tubes, pipet tips, disposable transfer pipets, a foam microtube holder, a DI water bottle, a sharpie to label, a hot water bath, Non-GMO food, Test food, and a centrifuge. Our materials for the setup of the PCR reactions included: PCR tubes, screwcap tubes, pipet tips, an aerosol barrier, a foam microtube holder, a PCR tube holder, a 2-20 microliter micropipette, an ice bath, a sharpie to label, a plastic bag, a styrofoam cup with ice water, and a black tray with ice water. Finally, the materials for the electrophoresis of the PCR products included: Agarose gel, PCR sample tubes from previous labs, PCR tube holders, running buffers (300-350 ml), loading dye, a PCR molecular weight
The light yellow precipitate was collected by suction filtration using a Hirsch funnel. The product was washed with two 1-mL portions of cold methanol followed by two 1-mL portions of diethyl ether. The product was dried in the oven at 110°C. The IR spectrum as a KBr pellet was obtained for the product and inosine for analysis.
After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After
The following results helped obtain the haplogroup that in which the sequence of mtDNA would identify. The PCR reaction worked, and this can be determined by looking at the agarose gel in figure 1. If the PCR reaction was successful, than a band should appear around 550bp. Individual AC displays a band around 550bp, this means the PCR reaction was successful. The band for individual AC, depicts a low concentration of product, because the band faint. After the purification process the concentration, A260/280 ratio, and A260/A230 ratio were determined by using the nanodrop. The concentration of mtDNA in the product was 60.9 ng/uL. The ratio for A260/280 was 1.79 and the ratio for A260/230 was 0.77. The A260 and 280 are a spectrometer measurement that measure absorbance at wavelengths of
PCR is the amplification of DNA by denaturing, annealing, and extension of a DNA template. Specific sequences can be amplified using single-stranded DNA that complements the target sequences known as primers. This process heats DNA until the strands separate, then primers bind to the target regions. DNA polymerase enzymes and single base nucleotides (dNTPs) are used to synthesize new strands of DNA to the target sequence. The end product will contain large quantities of the target sequence (Bean et al. 2015). The most notable in phylogenic studies is the 16S rRNA gene, because of it’s highly conserved primer-binding site and hyper variable regions that provide species-specific sequences within bacteria and archaea (Kolbert and Persin 1999). This gene is a component of the 30S small subunit of prokaryotic ribosome’s and serves as the primary site of protein synthesis. (Woese and Fox 1977). The 16S rRNA sequence can be amplified and matched to national databases provided by the National Center for Biotechnology Information (NCBI) using software termed Basic Local Alignment Search Tool (BLAST) to find regions of similarity between biological sequences for bacterial identification. Thus, providing a cost effective and timely method when compared to biochemical
Experiment 55 consists of devising a separation and purification scheme for a three component mixture. The overall objective is to isolate in pure form two of the three compounds. This was done using extraction, solubility, crystallization and vacuum filtration. The experiment was carried out two times, both of which were successful.
coli are picked and the plasmids are purified. The purified plasmids are used as a template for the sequencing reaction. The objective of the lab was to learn how to use the polymerase chain reaction (PCR) to amplify the small subunit ribosomal RNA gene from a bacteria colony, also be able to run an agarose gel to visualize the resulting PCR amplifications and extract the amplified DNA from the agarose gel.
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
In conclusion, this lab was successful in quantitative transfer and dilution although the final molarity might be slightly less than the calculated molarity. Therefore, to ensure the accuracy of the molarity in the final solution, techniques utilizing UV-Vis or conductivity measurement to determine the concentration of a colored or colorless solution need to be studied.