why RFLP can produce many bands on an electrophoresis gel and PCR (one set of primers) will only produce one or two bands on a gel for the same genome.
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Explain why RFLP can produce many bands on an electrophoresis gel and PCR (one set of primers) will only produce one or two bands on a gel for the same genome.
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- Explain why a positive control and negative control are included in PCR experiments. Explain the three steps involved in each cycle of polymerase chain reaction.Why is loading dye added to the DNA sample for gel electrophoresis? Explain the function of the following components in a PCR reaction:− Primer, dNTP, MgCl, Taq polymerase, buffer.Explain why DNA ladders are usually included during gel electrophoresis. One aspect of PCR that can be modified is the annealing temperature. In general, higher annealing temperatures show more specificity towards a single template, whereas lower annealing temperatures show less specificity and may bind to multiple regions throughout the genome. Discuss how using an annealing temperature that is too high or too low might influence the results of a PCR assay (and gel electrophoresis results) such as the one used in this study.A) What are the three main steps involved in PCR? Include temperatures and descriptions of each step. B) Explain why RFLP can produce many bands on an electrophoresis gel and PCR (one set of primers) will only produce one or two bands on a gel for the same genome.
- The final amount of each primer required in a PCR reaction is 25 picomol. If the total volume of the PCR reaction is equal to 100 µl and the stock concentration of each primer is equal to 0.0025 mM. Calculate the volume of stock primer that needs to be added in order to ensure a primer amount of 25 picomol.Our PCR samples already contain loading dye, but sometimes this isn’t the case. If your samples didn’t already contain dye and you wanted to load your PCR sample onto an agarose gel, you’d need to add loading dye to the proper concentration. There is a 6X loading dye available for use; how many µl of this loading dye will you add to 10 µl of your sample so that it is at a 1X working concentration? Show your work.Explain how the percentage efficiency of a real-time PCR reaction is determined using a theoretical experiment and why this is essential in any real-time PCR analysis.
- Explain the importance of different temperatures in PCR. Tm of forward primer= 62 °C Tm of reverse primer= 59 °C Gene size= 3 KbBelow is the result of PCR products running on agarose gel electrophoresis. You expect to see one band, but the results show two bands, as shown below. Why is there a lower band? What are they? How can we avoid such a band?You are performing PCR for the first time using some new primers that are 25 nucleotides in length with an estimated melting temperature of 65 0C to amplify a 200 nucleotide gene. After PCR, you run an agarose gel on the samples and observe a faint band at 25 nucleotides. What is the best possible explanation for the results? The primers hybridized to multiple sites on the DNA template. Too much template DNA was added to the PCR mixture. The primers dimerized preventing DNA transcription from occurring. A nuclease was in the solution causing degradation of the DNA.
- Describe the method of a PCR technique in which you can amplify fragments randomly? Briefly.You want to set up 50 µl total volume of a PCR reaction. You have a microfuge tube of Taq polymerase (5 units/ul), and each PCR reaction requires a final of 10 units of Taq polymerase. You have a microfuge tube of Taq Buffer (5X), and each PCR reaction requires a final of 1X Taq Buffer. How much of Taq polymerase and Taq buffer would you add? Select all that apply 10 ul of Taq buffer 2 ul of Taq polymerase 5 ul of Taq buffer 5 ul of Taq polymeraseIn PCR amplification Why is it important to know the length of the sequence you amplify?