NZYLong 2x Colourless Master Mix

This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 °C to 10 °C lower than Tm).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.
c) Template DNA damaged or degraded. An intact, high-quality template is essential to achieve a reliable amplification of large DNA fragments. Extreme care must be taken in the preparation and handling of DNA. Always use purified high-quality DNA as template.
d) Concentration of magnesium is too low. Mg²⁺ is included in the Master Mix at a final concentration of 2.5 mM, which is sufficient for most targets. For some targets, higher Mg²⁺ concentration may be required. Titrate from 2.5 mM to 4 mM (final concentration) in 0.5 mM increments. (Note: MgCl₂ is not provided in separate tubes).

Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences. Check the quality and concentration of primer solutions.  We recommend to prepare small-volume working aliquots from the stock solution.  Avoid multiple freeze cycles.

 The product should be stored at -20 ºC, in a constant temperature freezer.