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You are here : PCR / DNA AmplificationPolymerases ╗ one-fusion high-speed-fidelity Polymerase

one-fusion high-speed-fidelity Polymerase

HighEnd and exclusive Polymerase: faster speed, more accuracy, better results

 
 
 Cat.-no  Description  Amount  Price € Shop
 S450  one-fusion DNA Polymerase     200 units  105.00  add
 S455  one-fusion DNA Polymerase   1000 units  329.00  add

HighFidelity DNA Polymerase: One-fusion DNA Polymerase Datasheet


Features:
- Superior fidelity – about 50x improvement compared to Taq Polymerase
- Excellent performance across a wide range of “difficult” templates
- Long range amplification of complex targets - > 10 kb from genomic DNA
- High speed PCR - reduce reaction times
- dUTP poisoning resistance
- Resistance to blood containing DNA samples (up to 20 % of blood)

Applications:
- High Fidelity (Hi-Fi) PCR
- Cloning
- “Hi–Fi” - LD PCR
- “anticontaminated” PCR
- "direct blood" PCR

Description: One-Fusion DNA Polymerase is a unique artificial enzyme created on the basis of intellectual protein design planning by genetic engineering technique. The enzyme possess high fidelity feature. The processivity of the enzyme is very high, so the combination of processivity with fidelity results in dramatically increased yield of PCR products, very high sensitivity of PCR tests, ability to amplify “difficult” templates.

This dramatic increase in processivity results not only in shorter extension times, but also in more robust amplification and the ability to amplify long templates: really fast.

One-Fusion DNA Polymerase possesses the 5'->3' DNA polymerase activity, 3'->5' exonuclease activity and temperature-depended strand-displacement activity and generates blunt ends in the amplification products.

Unit definition: One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP's into acid-insoluble form in 30 minutes at 75°C under assay conditions: 25 mM TAPS-HCl, pH 9.0 (at 25°C), 100 mM KCl, 1.5 mM MgCl2 , 1 mM Beta-mercaptoethanol, 200 μM each dNTP, and 10 μg activated calf thymus DNA in 50 μl. 

Associated Activities: Endonuclease and exonuclease activities were not detectible after 2 and 1 hours incubation, respectively, of 1 µg lambda DNA and 0.22 µg of EcoR I digested lambda DNA, respectively, at 72°C in the presence of 15-20 units of One-Fusion DNA polymerase.

Storage buffer: 20mM Tris-HCl, pH 8.0, 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% Glycerol,0.5% Tween 20, stabilizers

Reaction buffers provided:
The product is supplied with 2,5x Reactionbuffer containing 3,75 mM MgCl2

Storage: store @ - 20°C

Transport: "blue ice" shipment

General protocol:
The optimal reaction conditions for One-Fusion DNA Polymerase may differ from PCR protocols for standard (Taq-like) DNA polymerases.
PCR conditions for one-Fusion DNA Polymerase is more similar in PCR conditions to “Phusion-like” DNA polymerases, e.g. the enzyme works better at elevated denaturation and annealing temperatures.
PCR reactions should be set up on ice. Prepare a master mix for the appropriate number of samples to be amplified.

Note! It is critical that the One-Fusion DNA polymerase is the last component added to the PCR mixture, since the enzyme exhibits 3'->5' exonuclease activity that can degrade primers in the absence of dNTPs.

Component

50µl reactions

25µl reactions

Final concentration

PCR grade Water

Up to 50 µl

Up to 25 µl

 

2.5x One-Fusion Buffer

20 µl

10 µl

1X

10 mM MIX dNTPs

1 µl

 0.5 µl

0.2 mM each

Primers

 

 

0.3-0.5 mM each

Template DNA

optionally

optionally

 

One-Fusion polymerase (2 U/µl)*

1 µl

0.5 µl

0.02 U/µL

* results in 1.5mM Mg2+, as the final concentration.
In some cases we recommend to optimize Mg concentration in the range 1.5-2.5mM´.

We recommend to use 50µl reaction for the PCR with One-Fusion polymerase 

Reaction components:

1. one-fusion Polymerase
An optimal amount of enzyme in 50µl reaction is 1U. In some cases it could be reduced up to 0.5U per reaction, depending on the length and complicity of amplified DNA sequence. For non-complex amplicons with the length less than 500bp and GC-content <60% amount of HF-Fuzz could be decreased up to 0.5-1.25U per 50mlreaction.

2. Buffer
2.5X Uni Buffers provides very high reproducibility across the wide range of amplification conditions, including “fast-PCR” (reduced time of PCR reaction). 2,5X Uni Buffer contains 1.5mM Mg2+, as the final concentration. In some cases we recommend to optimize Mg concentration in the range 1.5-4.5mM

3. DNTP’s
For most of applications 200mM of each of dNTP’s as final concentration is an optimal. It’s not necessary to optimize dNTP’s concentration. dUTP or other dUTP derivatives should be used replacing TTP in PCR reaction for “anti-contamination” PCR.

4. Primers
Usually 10-20pmol of each specific primer in reaction is enough to get good PCR result. If you are using 2-step PCR with the whole blood as a template, it’s better to use >= 20pmol of each primer.

5. PCR Additives
One-fusion polymerase is compatible with the most of commonly used PCR additives for enhancing of high GC-content DNA templates (glycerol, betaine, DMSO and other). If one will use any additives; take into account the changes of Tm of primers and DNA to correct annealing temperature.

Cycling:

 

 

Cycle step

2-step amplification

3-step amplification

 

Cycles

ToC

Time

ToC

Time

Initial Denaturation

98oC

1-5 min

98oC

1-5 min

1

Denaturation

Annealing

Extension

98oC

-

72oC

2-10 S

-

15-30 S/Kb

98oC

55-72°C *

72oC

2-10 S

10-30 S

15-30 S/Kb**

 

25-35

Final extension

72oC

4oC

1-2 min

hold

72oC

4oC

1-3 min

hold

1

 

 * optimal: Tm for the primer pair recommended as Tm of the lower primer, for the standard-oligos <20nt.
** For non-complex DNA templates (plasmid DNA, phage DNA, BAC clone) extension time could be reduced up to 15 sec/Kb

Further explanation to the protocol:

1. Denaturation:
a) Initial denaturation for 5 min at 98oC is necessary only for blood cells lysis;
b) for most applications, including “direct-blood” 5 sec at 98oC is enough for denaturation of the sample in during PCR run

NOTE: Do not use lower T denaturation, then 98oC; it can cause problems in PCR (nonspecific amplification, poor yield of PCR product, etc.)

2. Annealing/Extension:

For one-fusion DNA polymerase “Annealing” and “Extension” steps should be combined if:
-Tm of both primers are not differs dramatically (<3oC);
-Tm of the primers are >65oC (optimal Tm for the primers lays between 65-70oC)

If primers Tm is about 60-61oC for both primers ones can apply simple formula to determine starting Ta/e point - (Tm of the lower primer +72oC)/2. For most applications, it works fine.

To determine a better Ta/e run gradient amplification.

To avoid nonspecific band formation/smearing during amplification not exceed extension time of 30 seconds and use the highest ramp rate of amplificator ( the ramp rate >4-5oC preferable)

 

3. Post-PCR amplicon detection:

-         If you are using whole blood as a template, after amplification spine-down blood cells debris to avoid its application to the gel for DNA detection.

Note: Do not use excess of the blood (>10%), because the post-PCR debris volume of blood cells is very high, and does not allow to run more than 1-2 gels.

Recommendations:
- For complex DNA templates (human DNA) strongly recommended to apply Extension time as 30 sec/Kb for templates > 1, 5 Kb.

- For One-Fusion polymerase Tm of the primers should be corrected, as +3-5oC, comparing with Taq-based PCR conditions.

- For complex DNA templates (human DNA) strongly recommended to apply extension time as 30 sec/Kb 

related product:
SNPase for genotyping

>>> Service pages in English <<< >>> Serviceseiten auf Deutsch <<<
Copyrightę GeneOn 2007-15 print page: one-fusion high-speed-fidelity Polymerase
 


 
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Polymerases
 
one-Fusion high-speed-fidelity Polymerase
HighEnd and exclusive Polymerase: faster speed, more accuracy, better result
DFS Taq Polymerase DNA-free Discontinued
DFS Taq Polymerase (DNA free) is a highly purified polymerase (DNA-free) especially for PCR reactions where freedom from endogenous template (i.e., E. coli DNA) is essential
DFS-PLUS Taq Polymerase DNA-free
DFS PlusTaq Polymerase: more sensitivity; more processivity
H-SPlus-Taq DNA Polymerase
Hot Start Polymerase with ultra short inactivation time, free of MAB´s. The high concentrated versions are designed for Lyophilization
Hot-Start PCR: M-Superhot Taq Polymerase
Superior Taq Polymerase for Real Time PCR and Hot-Start PCR, low-copy number PCR, PCR of difficult templates, Hot-Start activity (only 5 min initial denaturation). The enzyme is developed to enhance the specificity, sensitivity and yield of DNA amplification. The enzyme provides a convenient setting up at room temperature
SNPase for SNP-Genotyping
Hot-Start Polymerase for SNP detection by allele-specific PCR and micro sequencing
Taq DNA Polymerase
Maximo Taq Polymerase is a highly purified polymerase for routine amplification
Taq DNA Polymerase 2X-preMix
Maximo Tag Polymerase as 2X-preMix includes all components (Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer) in an optimal concentration for routine PCR
Taq DNA Polymerase blue ready-to-load
Maximo Tag-Blue Polymerase is a highly purified polymerase for routine amplification. The enzyme is ready-to-load. No extra dye or loading buffer is needed!
m-Anti-Taq
Enhancer for Polymerase reactions
p-Taq Polymerase
p-Taq Polymerase is developed to enhance the specificity and yield of DNA amplification. The polymerase provides a convenient setting up at room temperature because of blocking the the enzyme with optimized mix of inhibitors
Bst DNA Polymerase
Bst DNA allows sequencing through problematic secondary structures
Pfu/Psp DNA Polymerase
Pfu DNA polymerase generated PCR fragments exhibits the lowest error rate of any thermostable DNA polymerases. The fragments are blunt-ended for direct ligation
Pfu/Psp red DNA Polymerase RTL
Pfu DNA polymerase (RTU=READY-TO-LOAD) is a convenient Mixture of proof-reading Enzyme, red dye and loading buffer. It generated PCR fragments exhibits the lowest error rate of any thermostable DNA polymerases. The fragements are blunt-ended for direct ligation
Pfu/Psp DNA Polymerase 2X-preMix
Pfu DNA polymerase 2X-preMix is a convenient Mixture of proof-reading Enzyme, reaction buffer dNTP´s and MgCl2. It generated PCR fragments exhibits the lowest error rate of any thermostable DNA polymerases. The fragements are blunt-ended for direct ligation
Tth DNA Polymerase
Tth DNA Polymerase Maximo is used to reverse-transcribe RNA efficiently in the presence of manganese. PCR can be performed in the same tube using the intrinsic DNA polymerase activity simply by chelation of manganese cation and the addition of magnesium.


 
 
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