NeoTaqII Master mix

Description

• NeoTaq II Master Mix: ready-to-use mix for robust and reliable standard PCR.
• Contains DNA polymerase, dNTPs, reaction buffer, and optimized additives.
• Supports amplification of DNA templates up to 6 kb.
• 2.5 mM MgCl₂ enables flexible PCR protocols.
• Blue and yellow tracking dyes allow direct loading onto agarose gels.
• No 3′→5′ exonuclease activity; produces A-overhangs suitable for TA cloning.
• For fluorescence/absorbance-based downstream applications, purify PCR products first or use the dye-free version.

Storage Conditions

This master mix should be stored at -85 °C to -15 °C in a freezer without defrost cycles to guarantee maximal shelf life. The high thermal stability of the enzyme mixture allows it to remain stable at 4 °C or even at roomtemperature for up to 4 weeks. The product will remain stable till the expiry date if stored as specified.

Quality control assays

Purity

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 be free of any detectable DNA contamination as evaluated through PCR. Thus, it is suitable for the amplification of bacterial and fungal DNA based on 16S and 18S rRNA PCR assays.

Nuclease assays

0.2-0.3 μg of plasmid DNA are incubated with NeoTaq II Master mix for 14-16 hours at 37 °C. Following incubation, the DNA is visualised on any Safe DNA Gel Stain stained agarose gel. There must be no visible nicking or cutting of the nucleic acid.

Functional assay

NeoTaq II Master mix is tested for performance in a polymerase chain reaction (PCR) for the amplification of different-sized DNA fragments (1 and 2.5 kb) from human genomic DNA. The resulting PCR products are visualised as single bands in any Safe DNA Gel stain stained agarose gel.

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 ° 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.

• Concentration of Mg2+ 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).

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.