Description
VeraSeq™ PCR Mix is a premixed, ready-to-use 2x solution containing VeraSeq™ 2.0 High-Fidelity DNA Polymerase (P7511L), dNTPS, MgCl 2 and reaction buffer at optimal concentrations to maximize the speed, accuracy and length of DNA synthesis. The formulation provides efficient, high-fidelity DNA amplification, cloning and synthetic biology applications.
Polymerase properties Storage temperature: –25°C to –15°C Extension rate: 15 second per kb at 72˚C Proofreading (3'→5' exo): Yes, strong Nick-translation (5'→3' exo): No Fidelity: >50x higher than Taq DNA Polymerase Strand displacement: No Thermostability: Highly thermostable Able to extend an RNA primer: No Extends from a nick: No Generate blunt end products: Yes Uracil read through: No Test Specification Functional Assay Amplification of 500 bp fragment from genomic DNA
Source of recombinant enzyme protein The protein is produced by a recombinant E. coli strain carrying the engineered VeraSeq™ 2.0 gene. Unit definition One unit is defined as the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid-insoluble form at 74°C in 30 minutes.
Protocol General precautions should be taken when setting up a PCR, including setting up the reaction on ice, adding master mix last, gently pipetting, thorough mixing and a quick centrifugation. The following procedure can be used as a guideline. Reactions may need to be optimized individually. Reaction setup (for 50 µL) Component Volume (µL) Final concentration Sterile H 2 O 20, variable N/A 2x VeraSeq PCR Mix 25 1x PCR Primer Cocktail 5 0.5 µM each Library DNA* Variable N/A * Total reaction volumes of library DNA and water should be adjusted to achieve a final reaction lolume of 50 µL. If the reaction volume needs to be >50 µL, the volume of the 2x Master Mix should be adjusted so that it constitutes 50% of the final reaction volume. Step Temperature Time Cycles Initial denaturation 98°C 30 seconds 1 Denaturation Annealing Extension 98°C 60°C 72°C 10 seconds 30 seconds 30 seconds To be determined by user † Final extension 72°C 4°C 300 seconds hold 1 * Cycling conditions may need to be optimized, depending on the amplicon of interest. † Number of cycles is dependent on the amount of input DNA and other specific sequence analysis requirements. Quality control analysis Functionality of 2x VeraSeq™ PCR Mix is assessed by its ability to amplify a 500 bp fragment from genomic DNA. Following PCR the 500 bp fragment was visualized by agarose-gel electrophoresis. Contamination Tests VeraSeq™ PCR Mix was tested prior to assembly and found to be free of contaminating endonucleases. Enzyme purity was >99% as determined by SDS-PAGE and negligible E. coli genomic DNA contamination was confirmed by qPCR. Specific activity was verified pre and post dilution.
