Description

• Hot-start NeoTaq II DNA polymerase is an engineered DNA polymerase with hot-start-like PCR activity.
• Limited activity at room temperature helps prevent nonspecific primer extension and primer-dimer formation.
• Delivers higher PCR specificity, sensitivity, and DNA yield.
• Activated in 5 minutes at 95°C for rapid reaction initiation.
• Supports amplification of DNA templates up to 6 kb with fast extension times of 15–30 s/kb.
• Requires minimal PCR optimization and allows room-temperature reaction setup.
• Hot-start NeoTaq II DNA polymerase lacks 3′→5′ exonuclease (proofreading) activity.
• Generates PCR products with 3′ A-overhangs, suitable for TA cloning with PCR cloning kits.

Storage conditions and reagents preparation

This product should be stored at -85 °C to -15 °C in a freezer without defrost cycles to guarantee maximal shelf life. The product will remain stable till the expiry date if stored as specified.

Quality control assay

Purity

Hot-start NeoTaq II DNA polymerase purity is > 90% as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie Blue staining.

Genomic DNA contamination

The product must comply with internal standards of DNA contamination as evaluated through real-time qPCR.

Nuclease assays

0.2-0.3 μg of plasmid DNA are incubated with 5 U of Hot-start NeoTaq II DNA polymerase, in 1× Reaction Buffer, for 14- 16 hours at 37 °C. Following incubation, the DNA is visualised on a Safe DNA Gel stainstained agarose gel. There must be no visible nicking or cutting of the nucleic acid. Similar tests are performed with Hot-start NeoTaq II buffer and MgCl2 solution.

Functional assay

Hot-start NeoTaq II DNA polymerase is extensively tested for performance in a polymerase chain reaction (PCR) of differentsized DNA fragments (1 and 2.5 kb) from human genomic DNA. The resulting PCR products are visualized as single bands in a any Safe DNA Gel stain-stained agarose gel. Similar functional tests are performed with Hot-start NeoTaq II buffer and MgCl2 solution.

Troubleshooting

No product amplification or low yield

• 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).

• 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.

• Additives required

Adding PCR-enhancing agents (5× Optimizer Solution or 2× GC-Enhancer Solution) may improve yield while allowing the amplification of difficult templates.

Presence of non-specific bands

• Non-specific annealing of primers

Adjust annealing conditions and/or design another set of primers, by increasing the length and avoiding complementary sequences.

• Mg2+ concentration is too high

Generally, 1.5-3 mM MgCl2, typically 2.5 mM final concentration, works well for the majority of PCR reactions. Optimal concentration depends on target template, buffer and dNTPs. Optimize magnesium concentration by supplementing MgCl2 in 0.5 increments up to 4 mM.