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First of all, the calculated primer melting temperature (Tm) and the size of PCR products should be similar so that all primers anneal at same temperatures during the PCR temperature cycling for better balance between PCR products in a multiplex reaction [41].
With the addition of calculated primer pair efficiencies, the ratio of gene expression for each target mRNA sequence between the two tissues was determined using equation (1).
Calculated primer annealing temperature (Tm) and expected product size in reference variety (i.e. M. acuminata 'Calcutta 4 and M. balbisiana "Pisang Klutuk Wulung" for mMaCIR102 to mMaCIR305 and M. acuminata 'Cavendish' for mMECIR0494 to mMECIR0500).
MISA search provides information about the type and localization of each individual microsatellite and parses the calculated primer sequences, their sequence and melting point, melting temperature, and expected PCR product size.
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To calculate primer amplification efficiency, a serial dilution of template DNA and a linear regression analysis of the resultant qPCR results were required.
The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides.
To calculate primer efficiency (E=10^(−1/m)), a dilution series was performed.
To cover the dynamic range of IL-2 and IL-6 an additional standard curve was made from a dilution series of a pool of cDNA samples showing high expression levels of IL-2 and IL-6 to calculate primer amplification efficiencies and correlations for these specific genes.
A dilution series of cDNA diluted 1, 10, 100 and 1000 times was run in each plate to provide a standard curve which was used to calculate primer efficiency to ensure efficiency between 1.8 and 2. Next a linear regression was applied to the standard curve with the subsequent formulas being used to interpolate the relative amount of the gene of interest in the samples [ 118].
MJOpticonMonitor3-Analysis software (version 3.1, Bio-Rad) calculates primers efficiency using the linear regression slope of the dilution series.
Alternatively, for primers containing mismatches, probabilities can be measured for individual primers and calculated for primer pairs.
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