DFS (DNA Free Sensitive) Taq DNA Polymerase is a thermostable DNA Polymerase of approximately 94 kDa isolated from eubacterium Thermus aquaticus strain YT-1. This unmodified Taq DNA Polymerase replicates DNA at 72°C. The DFS-Taq Polymerase catalyzes the polymerization of nucleotides into duplex DNA in 5’- 3’ direction in the presence of magnesium ions and possesses a 5’- 3’ exonuclease activity. The DNA-free Taq Polymerase is highly purified and is free of nonspecific endo- or exonucleases. Taq DNA Polymerase leaves single 3’-dA nucleotide overhangs on their reaction products.

Applications:

• Amplification of DNA fragments up to 5 kb

• Amplification of single-copy genes of eukaryotic genomes

• Labelling of PCR products with modified nucleotides (biotin-dUTP, fluorescein-dUTP)

• Cycle sequencing

• Generation of PCR product for TA cloning

• A valuable tool for PCR of bacterial genes due to the absence of contaminating bacterial DNA

Storage conditions:

-20°C

Concentration:

5 units/µl Supplied

Reaction buffers (10x):

Complete NH 4 Reaction buffer - Ammonium buffer with 25 mM MgCl 2

Incomplete NH 4 Reaction buffer Ammonium buffer without MgCl 2

Complete KCl Reaction buffer Potassium buffer 15 mM MgCl 2

Included MgCl 2 (100 mM)

We recommend Ammonium buffer for increased yield of PCR products and potassium buffer for increased specificity of PCR. MgCl 2 concentration should be optimized for each particular primer-template combination, meanwhile PCR is effective with 2.5 mM of MgCl 2 in the majority of cases.

Storage buffer:

10 mM K-phosphate, pH 7.4

0.1 mM EDTA

50% glycerol

0.1% Triton X-100

0.1% Tween 20

Recommended concentration of MgCl 2:

1.5 mM – 6 mM

Unit definition:

One unit of activity is the amount of enzyme required to incorporate 10 nmoles of dNTP into acidinsoluble DNA fraction in 30 minutes at 72°C.

Sensitivity:

The sensitivity of PCR reaction with DFS-Taq DNA Polymerase in optimal conditions is very high – in some reactions less than 6 DNA molecules were detected. The enzyme has a very good performance in single-copy gene PCR from genomic mammalian DNA.

Temperature stability, shelf life:

The enzyme has a half-life of 40 minutes at 95°C. After one year storage at -20°C no reduction of the activity was detected. Storage at ambient temperature for 30 days results in 30% reduction of activity.

• This Taq DNA Polymerase is absolutely free from bacterial DNA

• With BIORON buffer system for all purposes suitable

• High sensitivity: 6 molecules template are enough

• Efficient amplification of single copy genes

• High yields of products

Quality control tests

The purity is > 95% by SDS gel electrophoresis. The enzyme is tested on the absence of nonspecific endo- or exonucleases.

The following tests are performed with each lot of DFS-Taq DNA Polymerase:

• PCR with various templates – human and bovine genomic DNA, Phage Lambda DNA

• Endo/exonucleases contamination tests

• NO-TEMPLATE-TEST with the primers complementary to the conservative region of 16S bacterial ribosomal genes

• Storage test – 3 days at room temperature cause no change in performance. Taq DNA Polymerase of BIORON is free of DNA-contamination. No product can be detected when no-template PCR is performed with primers specific to the highly conserved region of 16S ribosomal RNA genes. In contrast to BIORON enzyme, Taq DNA Polymerases of a variety of suppliers contain E. coli DNA.