General description
The PCR Core Kit supplies all reagents (polymerase, buffers, dNTPs, etc.) for optimizing conventional PCRs of a specific template with maximum sensitivity, and specifcity. For convenience, each reagent component in the kit is supplied in a separate vial. This allows the user to easily change the amount of polymerase, dNTPs, and MgCl₂ in the reaction mix. Most importantly, the kit allows easy optimization of the Mg²⁺ concentration, one of the most crucial variables in the PCR.
Application
Principle
PCR is an in vitro method for enzymatically synthesizing defined sequences of DNA. The reaction uses two oligonucleotide primers that hybridize to opposite strands and flank the target DNA sequence to be amplified. A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by DNA polymerase results in the accumulation of a specific DNA fragment, the termini of which are defined by the 5′-ends of the primers.
Because the primer extension products synthesized in a given cycle can serve as a template in the next cycle, the number of target DNA copies approximately doubles every cycle; thus, 20 cycles of PCR yield about a million copies (220) of the target DNA. Primer elongation is catalyzed by Taq DNA Polymerase, a heat-stable DNA polymerase isolated from the thermophilic eubacterium Thermus aquaticus BM.
The PCR Core Kit is designed to determine individual PCR conditions for all basic PCR applications relying on Taq DNA Polymerase. Assays which might give better sensitivity and specificity with a non-standard Mg-concentration in the reaction buffer can be optimized using the Mg-free buffer supplied with this kit.
Packaging
1 kit containing 5 components.
Analysis Note
Each lot is PCR tested using λDNA. Each lot is also tested for the absence of exo- and endonucleases, and nicking activities according to the current Quality Control procedures.
Other Notes
Contents
Taq DNA Polymerase, 250 U, 5 U/μl in storage buffer: 20 mM Tris-HCl, 100 mM dithiothreitol, 0.1 mM EDTA, 0.5% Nonidet P40 (v/v), 0.5% Tween 20 (v/v), 50% glycerol (v/v), pH 8.0 (+4°C)
dNTP stock solution, 200 μl, containing each 10 mM dATP, dCTP, dGTP, dTTP, in sterile double-distilled water, pH 7.0
PCR reaction buffer, 10x concentrated, 2 x 1 ml, 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3 (+20°C)
25 mM MgCl2 stock solution, 1 ml
PCR reaction buffer without MgCl2, 10x concentrated, 1 ml, 100 mM Tris-HCl, 500 mM KCl, pH 8.3 (+20°C)
For life science research only. Not for use in diagnostic procedures.
Legal Information
Use of this product is covered by US Patent No. 6,127,155 and corresponding patent claims outside the US. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser′s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser′s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Human and veterinary diagnostic uses under Roche patent claims require a separate license from Roche. All uses other than internal research and human and veterinary diagnostic uses under Roche patent claims require a separate license from Thermo Fisher Scientific. Further information on purchasing licenses from Roche may be obtained by contacting the Licensing Department of Roche Molecular Systems, Inc., 4300 Hacienda Drive, Pleasanton, California 94588, USA or Roche Diagnostics GmbH, Sandhofer Strasse 116, 68305 Mannheim, Germany. Further information on purchasing licenses from Thermo Fisher Scientific may be obtained by contacting the Licensing Department of Thermo Fisher Scientific, 5791 Van Allen Way, Carlsbad, California 92008, USA.
