Difference: ProtocolsTaq (6 vs. 7)
Revision 72013-07-09 - AurkoDasgupta
Which polymerase is right for me?Types of polymeraseOur lab stocks two main types of polymerase:
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The following table lists a variety of polymerases, some immediately available in lab and some not, and useful information about each of them.
ApplicationsStandard Taq Diagnostic PCRSince Taq is the cheapest polymerase available, it is the most appropriate for diagnostic applications requiring multiple reactions, such as checking if your cloning or genomic insertion worked via PCR of the intended insert region. Colony PCR Taq is especially well-suited to PCR on the unpurified genome, as in colony PCR. Taq will more successfully (and less expensively) amplify a single product off a small number of cells than Phusion. Phusion High Fidelity High-fidelity PCR The error rate of Phusion DNA Polymerase is 4.4 x 10^-7; therefore, Phusion DNA Polymerase is suitable for all PCR applications requiring great accuracy. Cloning Phusion DNA Polymerase amplifies templates with an accuracy and speed previously unattainable with a single enzyme. This makes it a superior choice for cloning. Phusion DNA Polymerase possesses 5' - 3' polymerase activity and 3'-5' exonuclease activity and will generate blunt ended products. Specialized Cloning Techniques (Gibson, CPEC, SLIC, Overlap PCR) All of these specialized cloning techniques require that the polymerase being used does not have strand displacement or 5' → 3' exonuclease activity. As such, and given its speed and high fidelity, Phusion is an excellent choice for them. Long amplicons Extension and overall cycling times can be significantly reduced when using Phusion DNA Polymerase. Due to the high processivity, Phusion DNA Polymerase can amplify long templates in a fraction of the time other polymerases need. Anecdotally, amplification with Phusion tends to result in more nonspecific products than Taq, so it may not be suitable for amplifications of large fragments from genomic DNA. If you experience this problem, try switching polymerases. These two polymerases should meet most of your needs. However, other polymerases have unique advantages that may be useful in select circumstances Platinum Taq Very Stubborn Amplicons Anecdotally, Platinum Taq is something of a last resort polymerase. Ellington lab members have insisted that when no other polymerase will work, sometimes Platinum Taq will pull through and produce the desired product. KOD GC-rich Amplicons KOD has a very high success-rate amplifying GC-rich amplicons. It is also relatively inexpensive and fast.
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View topic | History: r8 < r7 < r6 < r5 | More topic actions...
