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.
Contributors to this topic
AurkoDasgupta, CraigBarnhart, JeffreyBarrick, MichaelHammerling
Topic revision: r7 - 2013-07-09 - 20:05:04 - Main.AurkoDasgupta
Lab.ProtocolsTaq moved from Lab.Taq on 2011-10-12 - 21:30 by Main.JeffreyBarrick -