DESCRIPTION
BioThermT™ is a modified BioTherm™ DNA Polymerase to facilitate incorporation of Biotin- and Digoxigenin-dUTP in DNA.

CONCENTRATION
5 units/µl

UNIT DEFINITION
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acidinsoluble form in 30 minutes at 72°C under the assay conditions (25 mM TAPS (tris-(hydroxy-methyl)-methyl-amino-propanesulfonic acid, sodium salt) pH 9.3 (at 25°C), 50 mM KCl, 2 mM MgCl₂, 1 mM ß-mercaptoethanol) and activated free of charge.

STORAGE BUFFER
10 mM K-phosphate buffer pH 7.0, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 0.01% Tween 20, 50% glycerol (v/v)

STORAGE TEMPERATURE
Store BioThermT™ DNA Polymerase below 0°C, preferably at -20°C, in a constant temperature freezer.

10X REACTION BUFER
160 mM (NH₄)₂SO₄, 670 mM Tris-HCl pH 8.8 (at 25°C), 15 mM MgCl₂, 0.1% Tween 20

Cat.No. GC-002-006 1.5 ml 10x reaction Buffer (contains MgCl₂)
Cat.No. GC-002-007 1.5 ml 10x reaction buffer without MgCl₂ plus 50mM MgCl₂ separately

ASSOCIATED ACTIVITIES
Endonuclease- and Exonuclease activities were not detectable after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoRI digested lambda DNA, respectively, at 72°C in the presence of 15-20 units BioThermT™ DNA Polymerase.

PRODUCT CITATIONS
Paetsch, M.; Mayland-Quellhorst, S. & Neuffer, B. (2006), Heredity 97(4), 283--290.

Figure 1. A 700 bp DNA fragment of a single copy gene was amplified without (lane 1) and with the addition of biotin-11-dUDP using different concentrations (lanes 2-4, MW = molecular weight standard). PCR reactions were run for 40 cycles, 10 sec at 92°C, 15 sec at 65°C, and 2 min at 72°C in a volume of 25 µl, including 10X amplification buffer (GeneCraft), 2 nM MgCl₂, 5 pmol of each forward and reverse primers, 1 ng of DNA, 1 unit of BioThermT Taq polymerase (GeneCraft) and 0.1 mM of each dNTPs, whereas dTTP was portionally substituted with increasing concentrations of biotin-11-dUTPs: 20 % (lane 2), 50% (lane 3), and 70% (lane 4). The incorporation of biotin-11-dUTP correlates with a size shifting of the amplified product. Note, the decrease of PCR efficiency at a proportion of 70% biotin-11-dUTP (lane 4) compared to the lower concentrations.

Figure 2. The efficiency of PCR mediated incorporation of biotin-11-dUDP into a DNA fragment of 900 bp was assesed quntitatively and by comparing different Taq polymerases using a ration of biotin-11-dUDP/dTTP of 1/1 (amplification conditions as described in picture 1). Lane 1 shows the unincorporated PCR product, lanes 2-4 show the biotinylated products using 0.1, 1, and 10 ng of starting DNA template and BioThermT polymerase (GeneCraft), lane 5 and 6 depicts biotnylated products using regular BioTherm polymerase (GeneCraft) and an enzyme from a competitor, respectively. Note, the template-dependent increase of PCR product yield, and the high efficiency of both BioTherm polymerases compared to a competitor's polymerase.

Figure 3. A 700 bp DNA fragment was internally labeled by PCR mediated incorporation of biotin-11-dUTP (biotin-11dUTP/dTTP = 1/4, 1/1, and 2.3, respectively; see picture 1). The PCR products were diluted to 500 pg, 50 pg, and 5 pg and dot blotted onto nylon membrane. The DNA was cross linked to the membrane by UV radiation and visualized by colorimetric detection by means of Streptavidin/alkaline phosphatase conjugate incubation in the presence of BCIP and XPhosphat for 3 hours at 37°C. Signal intensity was strongest at 1/1 concentration (50%) of biotin-11dUTP.