I'd suggest ignoring them and getting on with some experiments to determine optimal conditons. These calculators are not very accurate, and you need to verify the reactions in any case. NEBNext Index Oligo Selector This tool assists users in selecting barcode combinations when pooling NEBNext ® Multiplex Oligos for Illumina ®.Normally, primer concentrations in PCRs are in the range of 100-900 nM (I mostly use 300 nM for standard PCRs). Tm API A public Tm API allowing programmatic access to the NEB Tm calculator for incorporation in automated high throughput primer design workflows. NEBaseChanger can be used to design primers specific to the mutagenesis experiment. Fixed primers can be specified for the design of LAMP primers, and subsequent Loop primers are then designed based on LAMP primer selection. NEB LAMP Primer Design Tool can be used to design primers for your Loop-mediated Isothermal Amplification. The calculator also calculates the primer length, percentage of GC content, molecular weight, and extinction coefficient.Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products The method generates a large amount of raw numerical data and processing may notably influence final results PCR stands by your side in and out of the cathlab If extra bands are observed, higher annealing temperatures should be considered Note. 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. DescriptionQ5 ® High-Fidelity DNA Polymerase is a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme.

Neb tm calculator