Thermolase™-Thermostable DNA polymerase.
Thermolase™ (EC is a thermostable DNA-dependent DNA polymerase isolated from Thermus sp. and now available in a genetically engineered form. The enzyme has 5´-3´ polymerase (60-150 nt/s, about 1 kb/min), 5´-3´ exonuclease (strand displacement), 3´ terminal deoxynucleotidyl transferase actifity (which usualy results in addition of single dATP to the duplex DNA). Modified nucleotides (dNTPaS, c7GTP, biotin-11-dUTP, digoxigenin-11-dUTP and fluorescein-12-dUTP but not biotin-16-dUTP) are incorporated at high rates by the enzyme. Error rates are 1x10-4 for misincorporation and 2x10-5 for frameshift mutation. Only the last three bases adjacent to the 3´ end of the primer need to be correctly base paired, in order to initiate polymerization. The 5´ region of the primer is less sensitive to base mismatches. Therefore, new restriction sites can be easily introduced into an amplification product. Up to 9 kb can be amplified from lambda DNA and up to 5 kb from genomic DNA.
Unit definition

One unit of the enzyme will incorporate 10 nmol of dNTPs into an acid-insoluble form in 30 min at 72°C under standard assay conditions using a DNA template. Specific activity is 200.000U/mg protein.

Reaction buffers
B1 (x10, with Mg2+ ): 670 mM Tris-HCl, pH 8.8, 166 mM (NH4)2SO4, 15 mM MgCl2, 0.1% Tween 20.
Reaction conditions
For sample volume 20-100 µl use 200 mM dNTPs (end conc.), 25 pM (abs. quantity) each primer, 2 U of the enzyme, 100 ng genomic DNA as template. Addition of excess enzyme or template may lead to nonspecific amplification. For fragments <1 kb, please dilute the enzyme 1:5 with 1x buffer just before use.

Amplification conditions

For amplification of 3kb fragment please use 0,2 ml tubes and following conditions:
10x reaction buffer
2 mM dNTPs (200 µM each)
human genomic DNA (100 ng)
forward primer factor VIII.1(25 pM)
reverse primer factor VIII.4 (25 pM)
Thermolase™ (5 units/µl)
3 µl
3 µl
1 µl
1 µl
1 µl
0,5 µl
20,5 µl


30 µl



94° 3 min
58° 0,5 min
72° 4 min
93° 15 sec

Number of cycles

30 cycles


Store the control DNA at +4°C to avoid degradation by multiple freezing and thawing. Store all other reagents at -20°C.
PCR, mutation analysis (TaqMan™, PAMSA), site-directed mutagenesis, DNA labeling, 3´ A-tailing of blunt ends; primer extension; sequencing.

Quality control

activity, SDS-PAGE purity, absence of endo- and exonucleases, specific performance tests.


1. Chien, A. et al. (1976) J. Bacteriol. 127, 1550. 2. Kaledin, A.S. et al. (1980) Biokhimiya 45, 494. 3. Clark, J.M. (1988) Nucleic Acids Res. 18, 9677-9686.

Catalog #





Pack size

1000 U

2500 U





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