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Ulisses A flag Denunciar. A common logarithm, as will be used here, is defined as the exponent indicating the power to which 10 must be raised to produce a given number. An antilogarithm is found by reversing the process used to find the logarithm of a number; e.
Before this equation can be converted to a more useful form, two laws of logarithms must be stated, the product rule for logarithms and the power rule for logarithms. More discussions on logarithms can be found in Chapter 3.
Product Rule for Logarithms For any positive numbers M, N, and a where a is not equal to 1 , the logarithm of a product is the sum of the logarithms of the factors.
Problem 8. Solution 8. Placing these values into the equation gives the following relationship. Substitute the log values into the denominator and numerator. To find the log of a number on a calculator, enter that number and then press the log key. The initial target sequence was present in the reaction at 3 x copies. At the end of the 25 cycles, 4 x 10 a2 copies were produced. What was the efficiency E of the reaction?
Divide each side of the equation by 25 and simplify the left side of the equation. The Polymerase Chain Reaction Calculating the T m of the Target Sequence The T m melting temperature of double-stranded DNA is that temperature at which half of the molecule is in double-stranded conformation and half is in single-stranded form.
It doesn't matter where along the length of the molecule the single-stranded or double-stranded regions occur so long as half of the entire molecule is in single- stranded form. It is desirable to know the melting temperature of the amplicon to help ensure that an adequate denaturation temperature is chosen for thermal cycling, one that will provide an optimal amplification reaction.
DNA is called a polyanion because of the multiple negative charges along the sugar-phosphate backbone. The two strands of DNA, since they carry like charges, have a natural tendency to repel each other. In a water environment free of any ions, DNA will denature easily. In fact, under conditions of high salt, two strands of DNA can be made to anneal to each other even if they contain a number of mismatched bases between them. Most PCR reactions contain salt.
Potassium chloride KC1 is frequently added to a PCR reaction to enhance the activity of the polymerase. Magnesium is a cofactor of the DNA polymerase enzyme.
Other factors affect the T m of duplex DNA. Longer molecules, having more base pairs holding them together, require more energy to denature them. What is the amplicon's Tm? The M concentration of MgC12 is calculated as follows: 2.
Ulisses A flag Denunciar. There are several methods for calculating the Tm of the PCR primers. The concentrations of primer, dNTPs, magnesium, template, DNA polymer- ase and all other components can be determined by standard dimensional analysis. The polymerase chain reaction can be used to quantitate nucleic acids in a competitive reaction between an unknown template and a dilution series of known, calibrated template.