This FAQ covers the most common questions with regard to Optimase Polymerase.
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General Polymerase Questions: |
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What is Optimase Polymerase? |
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Optimase Polymerase is a novel proof reading polymerase isolated from a thermophilic Archaeal bacterium.
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What are the advantages of Optimase Polymerase versus the other polymerases on the market? |
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Optimase Polymerase provides 3’ to 5’ exonuclease activity unlike Taq polymerases. This proofreading function allows for much higher fidelity of amplification. Optimase Polymerase is also fully compatible with the WAVE® System DNASep® cartridges, whereas other proofreading polymerases and their buffers contain additives that shorten the lifetime of the DNASep cartridge. Optimase has been shown to have higher proof reading fidelity than other PCR high fidelity polymerases.
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Reaction and Amplification Questions: |
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Can I use Optimase Polymerase with all of my existing reaction and thermocycling conditions? |
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The kinetics of DNA amplification with a proof reading enzyme are different from a non-proof reader DNA polymerase. Therefore, the PCR conditions are different. Use ProtocolWriter™ at www.mutationdiscovery.com to calculate the correct thermocycler protocol.
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Do I have to use the formula provided to calculate the annealing temperatures of my primer sets? |
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The Quick Start Guide included with each kit gives a formula that has been used to develop the algorithm for ProtocolWriter. For your convenience use ProtocolWriter, which is easily accessible at www.mutationdiscovery.com when calculating the PCR conditions for your experiments. Further optimization can be performed if necessary. Annealing temperatures calculated using other formulas are typically lower than those obtained using the Quick Start Guide recommended formula, which can impact the fidelity of the reaction and result in lower specificity.
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Why are longer extensions times sometimes recommended for Optimase Polymerase? |
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Proofreading polymerases require time to remove misincorporated bases and replace with the correct nucleotide. For this reason Optimase Polymerase may require slightly longer extension times.
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Is it necessary to vortex the tube with the enzyme before performing the reactions as stated on the tube label? |
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Yes. It is recommended that the polymerase be vortexed for three seconds prior to addition to the reaction vials or master mix to ensure even distribution of the polymerase and storage buffer. Additionally after the addition of Optimase Polymerase, each reaction should be vortexed and briefly centrifuged prior to thermocycling.
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The enzyme was at room temperature (arrived with thawed ice in the box). Is it a problem? |
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Optimase Polymerase is very temperature stable, and most likely will still perform well. Our studies have shown that there is no problem. However, if you experience any difficulties, please contact Technical Support.
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Can I do Hot Start with Optimase Polymerase? |
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Optimase Polymerase is not a heat-activated polymerase, so there is no advantage to using a “hot start”. However, the polymerase activity will not be inhibited by most typical hot start protocols. |
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Can I use a buffer from another enzyme? |
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This is not recommended, especially when using with the WAVE System. |
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What additives for the buffer are compatible with Optimase Polymerase? |
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DMSO and betaine are compatible with Optimase Polymerase and the DNASep cartridges as long as the following guidelines are followed. DMSO up to 10% is compatible with the DNASep cartridge. Betaine is compatible with our cartridges provided recommendations in our cartridge warranty document are followed and with a maximum final concentration between 1.25 and 2.5M. Always use active clean and more frequent hot washes. |
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Can I add a Taq polymerase to Optimase? |
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Many researchers have successfully added Taq from various vendors to their PCR reactions with Optimase Polymerase. Please contact customer support for protocols or information. |
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Application Questions: |
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Are Optimase Polymerase PCR amplified products compatible with TA cloning? |
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Optimase Polymerase is a proof reading polymerase and it does not add A to the end of PCR amplified products in the same way as Taq polymerases. Therefore, Optimase Polymerase is more suitable for blunt-end cloning, rather than TA cloning. |
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ProtocolWriter Questions: |
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Can ProtocolWriter™ be used with other polymerases? |
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The algorithm has only been designed for Optimase Polymerase. Transgenomic does not guarantee successful use with other polymerases. Researchers have successfully used ProtocolWriter with GoTaq™ DNA Polymerase from Promega* and Taq DNA Polymerase from Sigma Aldrich. |
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*GoTaq™ DNA Polymerase is a trademark of Promega. |