Hot-start with high fidelity
BIO-21068 250 Units
BIO-21069 500 Units
Features
• High fidelity coupled with the specificity of hot-start
• Very high specificity and PCR sensitivity
• Convenient set up at room temperature
• Amplifies fragments =5Kb
• Available as a ready-to-use 2x reaction mix (AccuSure™ Mix)
Applications
• PCR requiring high specificity combined with high-fidelity
• Blunt-end cloning
• DHPLC compatible (detergent-free)
• Highly suited to site directed mutagenesis
Description
AccuSure™ is a heat-activated enzyme possessing 5’-3’ DNA polymerase and 3’-5’ proofreading exonuclease activities, offering extremely high fidelity and enhanced specificity. AccuSure is ideally suited for difficult PCR templates, such as DNA secondary structures and microsatellites. AccuSure produces blunt-ended amplicons of up to 5Kb in length.
AccuSure is optimized for use in a detergent-free system, making it suitable for direct loading of samples on to DHPLC columns.
PCR Reaction Conditions (for a 50µl reaction)
10x AccuBuffer 5µl
50mM MgCl2 Solution 0 - 2.0µl
100mM dNTP Mix (see below) 0.5-1.0µl
Template and primers as required
Enzyme 1-3µl
Water (ddH2O) up to 50µl
100mM dNTP Mix is available as a separate product (Cat. No: BIO-39028)
Activation step: Pre-heat at 95°C for 10 min.
Denature: 94-97°C;
Extension: 68°C allowing 1.5-2 minutes per Kb
Owing to AccuSure’s inherent 3'-5' exonuclease activity, the enzyme must be added last to a reaction in order to prevent primer damage.
This data is intended for use as a guide only; conditions will vary from reaction to reaction and may need optimisation.
General Considerations:
The enzyme must be activated by heat treatment before use. All reaction components (including AccuSure) should be added to the reaction, and then pre-incubated at 95ºC for 10 minutes. Subsequently, the reaction can be treated according to the user’s existing protocols for thermostable DNA polymerases.
The suggested final concentration of Mg2+ in the reaction is likely to be 2-4mM, but some optimization may be necessary to achieve the best possible results. Please, note that the reaction buffer already provides 2mM Mg2+. For first tests, use no less than 2.5 units of AccuSure in a 50µl reaction.
Product Insert
AccuSure™ DNA Polymerase
AccuSure is supplied with 10x reaction buffer containing MgSO4, which provides optimal final reaction conditions (1.5mM Mg2+) for most experiments. In order to allow optimization of reaction conditions, additional MgCl2 is provided.
The specificity and performance of AccuSure can be further improved with the use of 2x PolyMate Additive (not supplied, see associated products), which is designed for GC- or AT-rich DNA, ‘dirty; templates or sequences with a high level of secondary structure.
Product Specifications
Batch details:
Batch No: See vial
Units per vial: See vial
Concentration: 2.5u/µl
Components
AccuSure DNA Polymerase 250 Units 500 Units
AccuSure DNA Polymerase 100µl 200µl
10x AccuBuffer 1.2ml 2 x 1.2ml
50mM MgCl2 Solution 1.2ml 1.2ml
Reagent Specifications:
10x AccuBuffer: 600mM Tris-HCl, 60mM (NH4)2SO4, 100mM KCl, 20mM MgSO4, pH 8.3 at 25°C
MgCl 2 Stock Solution: 50mM MgCl2
Storage Buffer: 20mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1mM EDTA, 2mM DTT, 50% Glycerol and stabilizers.
Storage Conditions: AccuSure can be stored for 12 months at -20°C.
Shipping Conditions: On Dry Ice or Blue Ice
Unit Definition:
One unit is defined as the amount of enzyme that incorporates 10nmoles of dNTPs into acid- insoluble form in 30 minutes at 72°C.
Associated Products:
Product Name Pack Size Cat No
PolyMate Additive 2 x 1.2ml BIO-37041
ACCUZYME DNA Polymerase 250 Units BIO-21051
HyperLadder I 200 Lanes BIO-33025
Agarose 100g BIO-41026
SureClean Plus 1 x 5ml BIO-37047
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