Barrick Lab :: ProtocolsTaq

---+ Which polymerase is right for me?

---++ Types of polymerase
NEB polymerases may be purchased from the NEB freezer, which is located in the Biomedical Research Supply Core in MBB 1.316. An inventory of this freezer is available [[https://www.neb.com/en-us/nebnow/university-of-texas-austin-icmb-nebnow][here]].<br> 
Our lab stocks two main types of polymerase:  <br>
| *Type* | *Vender* | *Cost per unit* | *Product Information* |
| Taq DNA polymerase | NEB | $0.12 | [[https://www.neb.com/en-us/products/m0480-onetaq-dna-polymerase][link]] | 
| Phusion High Fidelity DNA polymerase | NEB | $0.97 | [[https://www.neb.com/en-us/products/m0530-phusion-high-fidelity-dna-polymerase][link]] | 

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<sup>–4</sup> to 10<sup>–5</sup> | 10<sup>–5</sup> to 10<sup>–6</sup> | 2.6x10<sup>–6</sup> | 1-3 x 10<sup>–6</sup> | 10<sup>–6</sup> | 10<sup>–6</sup> to 10<sup>–7</sup> |
| 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' &#8594; 5' exonuclease? (proofreading) | No | Yes (Pyrococcus) | Yes | Yes | Yes | Yes | 
| 5' &#8594; 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_ <br>
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_ <br>
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* <br>

_High-fidelity PCR_ <br>
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_ <br>
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)_ <br>
All of these specialized cloning techniques require that the polymerase being used does not have strand displacement or 5' &#8594; 3' exonuclease activity.  As such, and given its speed and high fidelity, Phusion is an excellent choice for them.

_Long amplicons_ <br>
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.

%RED%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. %ENDCOLOR%

*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_ <br>
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_ <br>
KOD has a very high success-rate amplifying GC-rich amplicons.  It is also relatively inexpensive and fast.
Barrick Lab > ProtocolList > ProtocolsTaq
Contributors to this topic
CraigBarnhart, AurkoDasgupta, JeffreyBarrick, MichaelHammerling, MeghnaVergis
Topic revision: r8 - 2025-03-20 - 21:27:28 - Main.MeghnaVergis
Lab.ProtocolsTaq moved from Lab.Taq on 2011-10-12 - 21:30 by Main.JeffreyBarrick -