PCR & DNA modifying enzymes

Description

Product Details

Enzyme is purified from an E. coli strain carrying a plasmid with Taq DNA polymerase gene from Thermus aquaticus YT-1. 

Reagents supplied: 10x Taq DNA polymerase Buffer with or w/o MgCl₂ and 25mM MgCl₂ 

Unit definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 72ºC. 

Taq DNA Polymerase is a thermostable enzyme that catalyzes 5’→3′ synthesis of DNA. The enzyme has no detectable 3’→5′ proofreading exonuclease activity, but possesses low 5’→3′ exonuclease activity.

PCR Guidelines:

PCR recommended reaction:

Components	20μl assay	50μl assay	Final Concentration/Quantity
10x Taq pol. buffer	2μl	5μl	1x
dNTP mix/10mM each	0.4μl	1μl	0.2mM each
25μΜ forward primer	0.4μl	1μl	0.5μM
25μΜ reverse primer	0.4μl	1μl	0.5μM
Template DNA	Variable	Variable	1-500ng
Taq DNA pol. (5u/μl)	0.1-0.2μl	0.25-0.5μl	1.25-2.5u / 50μl reaction
Sterile ultrapure water	Up to 20 μl	Up to 50 μl

PCR recommended conditions:

Step	Temperature	Time
Initial denaturation	94oC	2 min
25-35 cycles
Denaturation	94oC	45 sec
Annealing	45-68oC*	30 sec
Extension	72oC	1min/kb
Final extension	72oC	10 min
Hold	4oC	Indefinitely

*Annealing temperature depends on primer Tm

10x Taq pol. buffer: 500 mM KCl, 100 mM Tris-HCl pH 8.5 @ 25^oC, 15 mM MgCl₂, 1% Triton X-100

Storage conditions:

• 100 mM NaCl
• 50 mM Tris-HCl (pH 8.0 @ 25^oC)
• 1 mM DTT
• 0.1 mM EDTA
• 1% Triton X-100
• 50% glycerol
• Store at -20^oC

Standard DNA Polymerase Assay Conditions (not PCR conditions): The polymerase activity is assayed in 10 mM Tris-HCl (pH 8.5), 50 mM KCl, 1.5 mM MgCl₂, 0.1% Triton X-100, 200 μM each of dATP, dGTP, dCTP, dTTP (a mix of unlabeled and [³H] dTTP) and 12.5μg activated calf thymus DNA, in a final volume of 50 μl.

Functional Assay: Taq DNA Polymerase is tested for performance in the polymerase chain reaction (PCR) using 1.5 units of enzyme to amplify a 5000-bp region of Lambda DNA using 10ng as substrate. The resulting PCR product is visualized as a single band on an ethidium bromide-stained agarose gel.

Quality controls: Tested extensively for the absence of endo- and exodeoxyribonucleases. More details concerning quality controls could be found in Certificate of analysis.

Stability: Taq DNA polymerase is guaranteed to maintain stability for six months from the date of shipment when stored as directed.