Search results
Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products.
OligoAnalyzer™ Tool. Understand the expected properties of your oligos before you order them. Calculator for GC content, melting temperature (T m), molecular weight, extinction coefficient, µg/OD, nmol/OD, and more. Identify secondary structure potential. Minimize dimerization.
21 paź 2013 · Read this advice from our own thermodynamics specialist, Dr Richard Owczarzy, on the effects of probe and primer melting temperature (T m) on hybridization. He provides considerations for better oligo and PCR/qPCR assay design, including oligo concentration, salt, and base pairing mismatch positioning. Print Page.
Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm. The NEB Tm Calculator is recommended to calculate an appropriate annealing temperature. When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10). Extension:
24 sie 2015 · Step 2: Choose your design. The PrimerQuest Tool offers 4 design options that are based on algorithms specific for common experimental setups (Figure 1). By default, your results return the 5 best primer or assay designs. PCR (2 primers) qPCR (2 primers + probe; for use in 5′ nuclease assays) qPCR (2 primers; for use with intercalating dyes ...
To use this calculator select your DNA polymerase, type in or paste your primer sequences, and provide your final primer concentration. T m values, annealing temperature, and other data are automatically generated.
11 sty 2013 · The NEB Tm Calculator should be used to determine the annealing temperature when this enzyme. Typically, use a 10–30 second annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
