Source: Purified from an E. Coli strain carrying Thermus species DNA Polymerase overproducing plasmid.
Analysis condition: 25 mM Tris•HCl pH 9.0 at 25°C, 50mM KCl, 2 mM MgCl2, 0.1mg/ml gelatin, 200 µM each dATP, dGTP, dTTP, 100 µM [α32-P]dCTP (0.05µCi/nmol), 12.5 µg activated salmon sperm DNA.
Unit definition: One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nmol of dNTP into an acid-insoluble form in 30 minutes at 74ºC.
Storage & Dilution buffer: 50% glycerol (v/v), 20 mM Tris•HCl pH 8.7 at 25ºC, 100 mM KCl, 0.1 mM EDTA and stabilizers.
Quality control: The enzyme is free of nicking and priming activities, exonucleases and unspecific endonucleases. SDS/PAGE - 95 kD band, >98% pure. Activity and stability tested via thermocycling. The error rate per nucleotide per cycle is ~2.5x10-5; the accuracy is ~ 4x104. Estimated half life at 95ºC is 1.5 hours.
Reaction Buffers & Additives:
• 10x Reaction buffer D (Mg2+ free): 800mM Tris•HCl pH 9.4-9.5 at 25ºC, 200 mM (NH4)2SO4, 0.2% w/v Tween-20.
• 10x Reaction buffer DF (Mg2+ and detergent free): pH 9.4-9.5 800mM Tris•HCl, 200 mM (NH4)2SO4.
Routine storage of Reaction Buffers, 25 mM MgCl2 and additive at -20ºC is recommended.
ADDITIVE
10x Solution L facilitates amplification of difficult templates (for example, GC-rich DNA templates). This solution should be used at a defined working concentration (1x, 2x or 3x solution). Solution L IS NOT a reaction buffer and should be used ONLY IF non-specific amplifications occur.
Operating conditions: It is recommended to add the components in the following order: 10xbuffer, H2O, MgCl2 final concentration 2.0-2.5 mM (2.5mM is strongly recommended), dNTPs (200uM), DNA template (5ng-100ng/100µl), primers 30-100pmol/100µl) and finally the polymerase (2-5 U/100µl), for hard templates up to 10U/100ul.
IMPORTANT: To activate the polymerase, include an incubation step at 95ºC for 12 – 15 minutes at the beginning of the PCR cycle. Annealing temperature should be 2-6ºC lower than the primer melting temperature. Elongation time should be ~1 min/1 kb.
Shipping & Storage conditions:
Shipping and storage at room temperature for up to 1 month has no detrimental effects on the quality of Hot MultiTaq.
Routine storage: -20ºC .
Safety warnings and precautions: This product and its components should be handled only by persons trained in laboratory techniques. It is advisable that suitable protective clothing, such as laboratory overalls, safety glasses and gloves be worn. Care should be taken to avoid contact with skin or eyes. In case of contact wash immediately with water.
