Speedy NZYProof DNA polymerase

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 ° 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.

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

Speedy NZYProof DNA polymerase has 3´→ 5´ exonuclease activity and will generate blunt-ended products. So, blunt-end cloning is recommended, by using NZY-blunt PCR cloning kit (MB121) or other similar cloning kit. Nevertheless, if TA cloning is required, the generated blunt-ended PCR fragments can be modified by adding 3´A-overhangs with a different polymerase, such as Taq DNA polymerase (see the A-tailing protocol in the product brochure of NZY-A PCR cloning kit – MB053). Due to the strong proofreading activity of Speedy NZYProof DNA polymerase, we recommend immediately proceed with cloning after this procedure, in order to avoid lost of the 3´A-overhangs during storage. Alternatively, the PCR products can be cloned using the innovative NZYEasy Cloning & Expression Systems in a rapid, precisely and ligase-independent manner. For optimal cloning efficiencies, silica-column purification of the PCR product using NZYGelpure kit (MB011) or other similar kit is highly recommended. This procedure will remove dNTPs, unused primers and other impurities. Spin-column purification is sufficient as long as the product is >90% pure. Gel-extraction should be performed in case non-specific amplifications and/or primer-dimers are formed.