You are a researcher working in a molecular laboratory. One day, you need to run
ID: 146717 • Letter: Y
Question
You are a researcher working in a molecular laboratory. One day, you need to run a batch of PCRs. You assembled the reagents including Taq polymerase, made the reaction mixtures, and ran them in a thermocycler After the PCR program ran to completion, you performed a gel electrophoresis to checkthe quality of the PCR product. To your dismay, all reactions appeared to have failed! Nothingshowed up on the gel. You reviewed the thermocycler's log, which displayed the following information aboutyour recent PCR. cycles: : 89°C for 1 minute Denaturation Annealing; 50°C for 40 seconds Elongation: 65 °C for 3 minutes Can this temperature profile explain the PCR failure? Explain in your own words (4 points)Explanation / Answer
PCR is the technique used to amplify the DNA of a specific gene. PCR requires a DNA polymerase i.e Taq DNA polymerase which remains stable at very high temperature. In addition to this deoxyribonucleotides, primers are required.
There are three main stages of PCR reaction:
Denaturation: The double-stranded DNA is heated at high temperature to separate the two strands so that the separated strands can act as the template for new DNA strand synthesis.
Annealing: It is the where the temperature is lowered down a so that the primers get annealed to the DNA strands.
Elongation: The temperature is raised slightly so that DNA polymerase extends the new DNA strands.
The absence of DNA bands in the gel could be the following reason.
1. Here the number of PCR cycles are not mentioned, usually, there should be 25-30 cycles so that there should be enough amount of amplified DNA which can be visualized on the gel. A single cycle of PCR will not be able to make enough amplified DNA.
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