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Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products.
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. Use NCBI BLAST™.
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.
8 lis 2021 · Our Tm calculator accounts for specific buffer compositions that impact primer melting temps. The goal is to avoid weak thermodynamics that can cause primer dimers to form due to inefficient binding.
Recalculate primer Tm values using the NEB Tm calculator; Test an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair; Poor primer design: Check specific product literature for recommended primer design; Verify that primers are non-complementary, both internally and to each other; Increase length of primer ...
Whether you are creating an assembly that incorporates multiple fragments, conducting your entire PCR at a single temperature, or incorporating intentionally mismatched nucleotides, careful primer design can set you up for success before you even pick up your pipette.
Choose a DNA, RNA, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry.
