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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.
The following guide can be used to troubleshoot PCR reactions. Use our Tm calculator to help plan experiments and click here for optimization tips. Primer concentration can range from 0.05–1 µM in the reaction. Please see specific product literature for ideal conditions.
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.
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:
The calculator calculates recommended T m (melting temperature) of primers and PCR annealing temperature based on the primer pair sequence, primer concentration, and DNA polymerase used in PCR. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.
The annealing temperature (T a) chosen for PCR relies directly on length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the Tm of your primers. The optimal annealing temperature (T a Opt) for a given primer pair on a particular target can be calculated as follows: T a Opt = 0.3 x (T m of ...
