Thermolase™AB-Thermostable DNA polymerase for hot-start applications.
Description
Thermolase™AB (EC 2.7.7.7.) contains Thermolase™ DNA polymerase and polyclonal antibody to provide automatic hot-start PCR. The antibody is used to block polymerase activity during set-up of the PCR reactions at ambient temperatures. The inhibition of DNA polymerase is completely reversed when the temperature is raised above 70°C. At the first template denaturation step in thermal cycling, the enzyme-antibody complex dissociates and the antibody is rendered nonfunctional. At the same time, the activity of DNA polymerase is restored, and the enzyme functions normally during the course of the PCR.
 
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.

Amplification conditions

For amplification of 1,5 kb 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.3(25 pM)
reverse primer factor VIII.4 (25 pM)
Thermolase™AB (5 units/µl)
H2O
 
 
3 µl
3 µl
1 µl
1 µl
1 µl
0,5 µl
20,5 µl

total

30 µl

Hot-start

Steps

94° 3 min
 
58° 0,5 min
72° 2 min
93° 15 sec

Number of cycles

30 cycles

 

Storage
Store the control DNA at +4°C to avoid degradation by multiple freezing and thawing. Store all other reagents at -20°C.
Applications
Hot-start protocol provides higher specificity and minimizes background. It is applied for amplification of complex cDNA templates, low-copy targets or in multiplex PCR.

Quality control

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

References

 

Catalog #
T16.4

T16.5

 

 

 

Pack size

1000 U

2500 U

 

 

 

 

Copyright © 2002 M.B. Enzymes GmbH