Which polymerase is right for me?

Types of polymerase

Our lab stocks two main types of polymerase:
Type Vender Cost per unit Product Information Image
Taq DNA polymerase NEB $0.12 link
Standard Taq
Phusion High Fidelity DNA polymerase NEB $0.97 link
Phusion HF

The following table lists a variety of polymerases, some immediately available in lab and some not, and useful information about each of them.

Taq Platinum Taq KOD PFU Vent Phusion
Species Thermophilis Aquaticus Mix of Taq, Pyrococcus species GB-D polymerase and Platinum® Taq Antibody Thermococcus kodakaraensis Pyrococcus furiosis Thermococcus litoralis Pyrococcus + Processivity domain
Fidelity (err/bp) 10–4 to 10–5 10–5 to 10–6 2.6x10–6 1-3 x 10–6 10–6 10–6 to 10–7
Elongation Rate 1 min/kb 1 min/kb 20-30 sec/kb 1 min/kb 1 min/kb 20-30 sec/kb
Processivity ~ 50 bases > 50 bases >300 bases 20 ? 35
Nicking Activity None None None ? < 10% ?
Amplicon Size < 5kbp Up to 20 kbp Plasmid/Linear = 6kbp Genome = 2kbp Vector: Up to 15 kbp Genome = Up to 19 kbp ? Genome = 10 kbp Plasmid/Linear = 20 kbp
3' → 5' exonuclease? (proofreading) No Yes (Pyrococcus) Yes Yes Yes Yes
5' → 3' exonuclease? Yes Yes No No No No
Strand displacing? Yes Yes No No Yes No
Overhang? 3' - A 3' - A No No No No
GC-Rich Performance Low Low High Medium High Medium
Hot Start? No Yes Available Available No Available

Applications

Standard Taq

Diagnostic PCR
Since 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.

 Barrick Lab  >  ProtocolList  >  ProtocolsTaq

Topic revision: r7 - 09 Jul 2013 - 20:05:04 - Main.AurkoDasgupta
Lab.ProtocolsTaq moved from Lab.Taq on 12 Oct 2011 - 21:30 by Main.JeffreyBarrick - put it back
 
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