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Description
A robust, efficient proofreading enzyme that gives increased fidelity in high-yield PCR, for use in all routine cloning applications.
Product Highlights
Efficient – highly productive target amplification and removal of 3’ A overhangs
Accurate – possesses 3’ - 5’ proofreading exonuclease activity that delivers an error rate of 3.0 x 10-6 for increased PCR fidelity versus Taq DNA polymerase
Sensitive – high-yield amplification from limiting amounts of human, animal and plant template DNA
Robust – developed for reliable amplification of even the most challenging targets, including genomic DNA and GC-rich targets
Flexible – ideal for amplifying any target up to 5 kb from DNA extracted from mammalian tissue samples
Convenient – advanced buffering system minimizes the requirements for PCR optimization, thereby reducing time-to-results and eliminating the cost of unnecessary repeats
Simple – mix reduces pipetting burden for increased consistency in assay set-up and reduced contamination risk
Product Description
ACCUZYME™ is a proprietary proofreading enzyme that offers increased-fidelity and high PCR yield, even in demanding applications. ACCUZYME has an error-rate of 3.0 x 10-6 and results in blunt-ended amplicons up to 5 kb in length, making it ideal for use in cloning and site-directed mutagenesis.
ACCUZYME is supplied with a buffering system that provides ideal conditions for most PCR assays. Consequently, the cost and effort typically associated with optimizing assay performance is often eliminated. In circumstances where further optimization is required to improve PCR specificity and/or yield, ACCUZYME includes an additional vial of MgCl2.
ACCUZYME Mix dramatically reduces the time needed to set up reactions, thereby minimizing the risk of contamination. Greater efficiency and reproducibility are achieved by reducing the number of pipetting steps that often lead to variation in reaction set-up.
An 800 bp fragment was amplified from 500 ng, 50 ng, 5 ng, 0.5 ng, 50 pg and 5 pg (lanes 1-6 respectively) of human genomic DNA using ACCUZYME. HyperLadder 1kb (M). The result illustrates that ACCUZYME gives high product yield even from limiting amounts of template.
All components are shipped on dry/blue ice and should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided. When stored under the recommended conditions and handled correctly, full activity of the reagents is retained until the expiry date on the outer box label.
Components may also be stored at +4°C if required, although storage at -20°C is recommended. When stored at +4°C, reagents will remain stable for a period of 2 weeks from date of receipt.
At Meridian we pride ourselves in supplying high-quality polymerases to suit your requirements. To aid your selection of the most suited enzyme for your specific applications, please see our enzyme selection tool.
All our polymerases are guaranteed for a period of 12 months from the date of purchase. These should be stored at -20°C during this time for optimal retention of activity.
Please Note: We do not recommend the storage of our polymerases at -80 °C as ice crystals could form on the active site, which may affect or destroy the activity of the enzyme.
PCR can be a challenging technique, with various parameters to optimize to achieve the best results. If you are having problems, these could be easily resolved by addressing a few issues. Please see our PCR troubleshooting guide for suggestions and help with your specific problems.
Observation
Recommended Solution(s)
No or low PCR yield
Enzyme concentration too low – increase the amount of enzyme in 0.5 U increments.
Primers degraded – check quality and age of the primers.
Magnesium concentration too low – increase concentration in 0.25 mM increments with a starting concentration of 1.75 mM.
Primer concentration not optimized. Titrate primer concentration (0.3-1 µM); ensuring that both primers have the same concentration.
Template concentration too low – Increase concentration of template.
Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated.
Multiple Bands
Primer annealing temperature too low. Increase annealing temperature. Primer annealing should be at least 5°C below the calculated Tm of primers.
Prepare master mixes on ice or use a heat-activated polymerase.
For problems with low specificity. Try adding 3% DMSO (not supplied) to improve specificity.
Smearing or artifacts
Template concentration too high. Prepare serial dilutions of template.
Too many cycles. Reduce the cycle number by 3-5 to remove non-specific bands.
Enzyme concentration too high - decrease the amount of enzyme in 0.5 U increments.
Extension time too long. Reduce extension time in 0.5-1 minute increments.
These terms refer to parameters to be considered when performing PCR and are important features in selecting the correct enzyme for your needs. Understanding what they mean is therefore crucial:
Yield: The amount of DNA produced in a PCR reaction.
Fidelity: The accuracy of the enzyme at incorporating the correct dNTP to the elongating DNA strand.
Processivity: The length of time a polymerase is associated with the template and therefore the size of fragment which can be amplified.
Specificity: A measure of the unwanted by-products generated in a reaction.
In nature, the ability of a DNA polymerase to correct misincorporations of nucleotides in the DNA strand being elongated is often crucial to the survival of the host organism. This ability is termed "proofreading activity" and occurs in the 3' to 5' direction. This activity also leads to the polymerase removing unpaired nucleotides overhanging at the 3'end (A-overhangs), creating blunt ends.
All Meridian polymerases are available in the convenient format of a 2x master mix, containing all the reagents and additives necessary to perform successful PCR, with the exception of template, primers and water.
This formulation not only provides a convenient and hassle-free PCR setup, but also significantly reduces the chances of human error, inaccuracies and contamination by reducing the pipetting steps required.
All Meridian PCR mixes come supplied at a 2x concentration, requiring the user simply to add template, primers and PCR grade water to a final concentration of 1x.
We recommend a final reaction volume of 50 µL, this requiring the use of 25 µL of the 2x master mix and to make up to 50 µL using template, primers and PCR grade water.
Whilst the exact composition of the mixes is proprietary information, all mixes are made up of reaction buffer, magnesium, dNTPs, polymerase and additives, designed to keep the polymerase stable in the mix, as well as provide the optimal conditions for PCR.
Meridian's mixes contain magnesium chloride at the following concentrations (please see table below). An additional tube of 50 mM MgCl2 solution is supplied with each mix to facilitate further optimization if required.
If possible, we would recommend an additional cleanup of affected samples, for instance, customers had very good experiences with the clean-up of samples containing humic acids with SureClean Plus. Alternatively decreasing the template concentration will help to minimize the amount of inhibitors. The amount of primer can be increased, but do not exceed the suggested limits.
During PCR setup at room temperature, standard polymerases will have some activity. When using a hot-start polymerase, this enzyme will have no activity at room temperature, thus reducing the risk of unspecific products in the reaction due to these mis-primed oligonucleotides. This feature makes this type of polymerase ideally suited to high-throughput applications, where the reactions may be left sitting at room temperature for prolonged periods of time.
ACCUZYME DNA polymerase has an extremely fast extension rate compared to other proofreading enzymes, which are naturally slower than non-proofreading polymerases due to their ability to go back and correct nucleotide mis-incorporations. These polymerases will extend at a rate of as little as 30 s/kb, depending on the template amplified. Extension should be carried out at 68°C for optimal results.
