(a)
To determine: The process which occurs at a temperature range of 92-95 °C in a PCR reaction.
Introduction: The polymerase chain reaction is a method used to produce multiple copies of a specific DNA segment. Thousands to millions copies of a specific DNA fragment are produced by using PCR.
(b)
To determine: The process which occurs at a temperature range of 45-65 °C in a PCR reaction.
Introduction: The PCR has two limitations; to synthesize primers; some information about the
(c)
To determine: The process which occurs at a temperature range of 65-75 °C in PCR reaction.
Introduction: The PCR has many advantages such as it is an important tool for detecting bacteria and viruses. The technique is advantageous for studying the samples from single cells, fossils, and the crime scene.
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Concepts of Genetics (12th Edition)
- 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.arrow_forwardYour amplicon from PCR was subjected to AGE for analysis. You are shown the image of the gel loaded with the following lanes: (A) negative control, (B) size ladder (1, 2, 3, 4, 6, and 10 kb), (C) gDNA extract, (D) PCR amplicon. However, due to mishaps while loading the gel with the samples, you are not sure which lane is which. You are shown a diagram of the obtained gel below. a. Label each lane of the gel. Write only the corresponding letters in the wells above. b. Above each band in the size ladder, write its size (in kb). c. Approximate the size (in kb) of the polystyrenase gene. Write your answer above the band corresponding to the gene. Bonus: If you wish to identify the bacterial species in this scenario, what gene is most commonly and routinely sequenced? Answer: __________________________________arrow_forwarda) (iii) (iv) Polymerase chain reaction (PCR) is a technique that enables multiplication of specific DNA sample at minute amount to millions or billions of copies at a short time span. (i) Figure 1 indicates the chemicals added into the PCR reaction tube prior to the addition of thermostable DNA polymerase. Do you agree with the list? Justify your answer. DNA template Buffer (containing Tris-HCI, KCI, Mg2+) ddNTP Forward primer Reverse primer Figure 1 If TA cloning is planned to be carried out after amplification of the gene, which thermostable DNA polymerase will you select and the reason for your selection? Why is the optimal annealing temperature vital in this technique? Explain how this temperature (too high or too low) will affect the efficiency of this reaction. If the primers you purchased possessed the following information: Number of Guanine: 4 Number of Adenine: 3 Number of Thymine: 4 Number of Cytosine: 5 Calculate the melting temperature of this primer and estimate the…arrow_forward
- There are many PCR techniques available to suit the needs of all researchers in their laboratory task. (i) (ii) What is the major difference in the functions performed by the conventional PCR and real time PCR? Shania is planning to study the gene expression of Escherichia coli after a drug- treatment. She needs to decide between two types of chemistries to detect her PCR products (TaqMan Chemistry vs. SYBR Chemistry) using real-time PCR instruments. Compare and contrast between TaqMan Chemistry and SYBR Chemistry.arrow_forwarda) The neomycin resistant colonies were screened by PCR using the primers A and B. Only seven of the 515 colonies (AB1 – AB7) resulted in the production of a PCR product of the expected size. The remaining 508 colonies failed to produce a PCR product (of any size). Explain why this would have been observed. Note: there is a rational explanation for this other than “the experiment did not work or there was a problem with the reagents”.arrow_forward1) Prepare the following enzymatic reaction, present it in tabulated form. In a final volume of 30 ul, where buffer 4 (10 ml). How much volume of each reagent would be used and how much of water? Is there any problem? 2) The DNA pol 1 enzyme comes at a concentration of 50,000 U/ml. You have to prepare a 50 ug PCR reaction where you must use 0.05 U/ml reaction. You add 10 ul of PCR buffer, 2 ng of tempered DNA that is at a concentration of 0.5 ng/ul, primers (which are at 200 mM) so that each one remains at a concentration of 200 uM, Mg+2 that is 5 mM (10 X), enzyme and water. Present the table of all the reagents included in the reaction, the volumes of each one in ul. Present where the initial and final concentration of each reagent applies. Assume you have micropipettes for all values.arrow_forward
- You are working in a molecular biology laboratory and are having challenges with your PCR. You decide to double-check the PCR protocol programmed into the thermal cycler and discover that the annealing temperature was programmed to be 65 °C instead of 50 °C, as you had intended. What effects would this mistake have on the PCR reaction? Suggest potential solution(s).arrow_forwardA) 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.arrow_forwardYou have a 100 micromolar stock solution of dNTPs in the freezer and are setting up a PCR reaction in which you need to have a final concentration of 20 micromolar dNTPs. In a reaction volume of 25 microliters, how much of your stock dNTPs do you add to each PCR reaction?arrow_forward
- You are setting up your PCR reaction and accidentally add twice as much of the salt buffer as you were supposed to. Select all that apply. 1. How will this impact product formation? 2. In what way(s) will the reaction be altered? (a) ...because primer/template binding will be altered (b) ...because the mechanism of dNTP addition will be altered. (c) You will get more of the desired PCR product... (d) ...because template denaturation will be altered. (e) You will get the same amount of the desired PCR product... (f) You will get less of the desired PCR product...arrow_forwardIn PCR amplification reaction, 5 mM EDTA was erroneously added to your final reaction, and you did not get any products. What is a possible explanation for this (based on the properties of EDTA)?arrow_forwardWhat would be the final primer concentration if 0.5 μl of 10 μM primers were added to a PCR reaction with a final volume of 20 μl?arrow_forward
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