Ultra HiFidelity PCR Kit

High fidelity, high specificity and high efficiency hot-start PCR premix.

Ultra HiFidelity PCR Kit is a new high-fidelity PCR amplification premix suitable for PCR-related cloning and detection. The Ultra HiFi DNA Polymerase contained in the kit is a new fast and high-fidelity DNA polymerase developed by directed molecular evolution technology. It enhances the affinity of DNA polymerase to templates, improves the amplification speed and extension ability of the enzyme, and increases the success rate of PCR and product yield.

Cat. No Packing Size
4992970 1ml
4992971 5*1ml
4992978 5*5*1ml

 

 


Product Detail

Product Tags

Features

■ Easy to operate: This kit is supplied as 2× premix, and PCR can be performed by simply adding templates and primers.
■ High fidelity: The fidelity is 50 times that of Taq polymerase.
■ High specificity: Excellent hot-start performance to ensure product specificity..
■ Rapid amplification: The extension speed can reach 10-15 sec/kb.
■ Strong extensibility: Up to 20 kb DNA fragments can be amplified.
■ Wide applicability: The kit contains PCR Enhancer and is suitable for the amplification of high GC and complex templates.

Specification

Type: High fidelity DNA Polymerase
Amplification speed: 10-15 sec/kb
Fragment size: <20kb
Applications: High fidelity PCR amplification, gene cloning, high GC template amplification, gene cloning of complex genomes, cDNA high fidelity amplification, SNP detection, site-specific mutation, etc.
DNA Extraction Yield from Various Plant Tissues:
Note: DNA yield depends on sample types. All the materials above are from tender leaves.

All the products can be customized for ODM/OEM. For details, please click Customized Service(ODM/OEM)

Experimental Example

Experimental Example Hot-start to ensure product specificity
Figure 1. Ultra HiFi has excellent hot-start function to ensure the specificity of amplification products. Molecular beacons method was applied (Ma et al., Anal Biochem, 2006).
Experimental Example Excellent high fidelity, 50 times higher than Taq Polymerase
Figure 2. The fidelity of Ultra HiFi is 50 times higher than that of common Taq polymerase. The polymerization fidelity of Taq polymerase (without corrective activity) is used as reference.
Experimental Example Rapid amplification and long fragments can be amplified fast
Fig. 3. Ultra HiFi can extend up to 5 sec/kb for fragments smaller than 4 kb. For long fragments, the amplification time can be extended appropriately. For fragments more than 15 kb, the extent speed can be up to 30 sec/kb. M: TIANGEN D15000 Marker
Experimental Example Experimental Example Experimental Example Strong universality and high specificity, easy to read high GC and long fragments from different sources
Figure 4. Ultra HiFi has high specificity to ensure amplification success rate and product quantity for different types of templates.
A. Ultra HiFi amplification results
B. Hi-Fi enzyme amplification results of Supplier K
C. Hi-Fi enzyme amplification results of Supplier N
M: TIANGEN D15000 Marker
Lane 1-5. Amplification results of templates with different length: 1. 750 bp; 2. 1 kb; 3.
2 kb; 4. 4 kb; 5. 6 kb
Lane 6. Amplification result of high GC template: 1915 bp (GC%: 70%);
Lane 7-11. Amplification result of 2 kb templates from various genome: 7. Rat; 8.
Rice; 9. Wheat; 10. Corn; 11. Bacteria;
Lane 12-14. 8 kb long fragment amplification result: 12. Rice; 13. Corn;

FAQ

Q: No amplification bands

A-1  Template

■ The template contains protein impurities or Taq inhibitors, etc. ——Purify DNA template, remove protein impurities or extract template DNA with purification kits.

■ The denaturation of template is not complete ——Appropriately increase denaturation temperature and prolong denaturation time.

■ Template degradation ——Re-prepare the template.

A-2  Primer

■ Poor quality of primers ——Re-synthesize the primer.

■ Primer degradation ——Aliquot the high concentration primers into small volume for preservation. Avoid multiple freezing and thawing or long-term 4°C cryopreserved.

■ Inproper design of primers (e.g. primer length not sufficient, dimer formed between primers, etc.) -Redesign primers (avoid formation of primer dimer and secondary structure)

A-3   Mg2+concentration

■ Mg2+ concentration is too low ——Properly increase Mg2+ concentration: Optimize the Mg2+ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg2+ concentration for each template and primer.

A-4   Annealing temperature

■ The high annealing temperature affects the binding of primer and template. ——Reduce the annealing temperature and optimize the condition with a gradient of 2°C.

A-5   Extension time

■ Short extension time——Increase extension time.

Q: False positive

Phenomena: Negative samples also show the target sequence bands.

A-1   Contamination of PCR

■ Cross contamination of target sequence or amplification products ——Carefully not to pipet the sample containing target sequence in the negative sample or spill them out of the centrifuge tube. The reagents or equipment should be autoclaved to eliminate existing nucleic acids, and the existence of contamination should be determined through negative control experiments.

■ Reagent contamination ——Aliquot the reagents and store at low temperature.

A-2   Primer

■ Mg2+ concentration is too low ——Properly increase Mg2+ concentration: Optimize the Mg2+ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg2+ concentration for each template and primer.

■ Improper primer design, and the target sequence has homology with the non-target sequence. ——Re-design primers.

Q: Non-specific amplification

Phenomena: The PCR amplification bands are inconsistent with the expected size, either large or small, or sometimes both specific amplification bands and non-specific amplification bands occur.

A-1  Primer

■ Poor primer specificity

——Re-design primer.

■ The primer concentration is too high ——Properly increase the denaturation temperature and prolong denaturation time.

A-2   Mg2+ concentration

■ The Mg2+ concentration is too high ——Properly reduce the Mg2+ concentration: Optimize the Mg2+ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg2+ concentration for each template and primer.

A-3   Thermostable polymerase

■ Excessive enzyme amount ——Reduce enzyme amount appropriately at intervals of 0.5 U.

A-4   Annealing temperature

■ The annealing temperature is too low ——Appropriately increase the annealing temperature or adopt the two-stage annealing method

A-5   PCR cycles

■ Too many PCR cycles ——Reduce the number of PCR cycles.

Q: Patchy or smear bands

A-1  Primer ——Poor specificity ——Re-design the primer, change the position and length of the primer to enhance its specificity; or perform nested PCR.

A-2  Template DNA

——The template is not pure ——Purify the template or extract DNA with purification kits.

A-3  Mg2+ concentration

——Mg2+ concentration is too high ——Properly reduce Mg2+ concentration: Optimize the Mg2+ concentration by a series of reactions from 1 mM to 3 mM with an interval of 0.5 mM to determine the optimal Mg2+ concentration for each template and primer.

A-4  dNTP

——The concentration of dNTPs are too high ——Reduce the concentration of dNTP appropriately

A-5  Annealing temperature

——Too low annealing temperature ——Appropriately increase the annealing temperature

A-6  Cycles

——Too many cycles ——Optimize the cycle number

Q: How much template DNA should be added in a 50 μl PCR reaction system?
ytry
Q: How to amplify long fragments?

The first step is to choose the appropriate polymerase. Regular Taq polymerase cannot proofread due to lack of 3’-5’ exonuclease activity, and mismatch will greatly reduce the extension efficiency of fragments. Therefore, regular Taq polymerase cannot effectively amplify target fragments larger than 5 kb. Taq polymerase with special modification or other high fidelity polymerase should be selected to improve extension efficiency and meet the needs of long fragment amplification. In addition, the amplification of long fragments also requires corresponding adjustment of primer design, denaturation time, extension time, buffer pH, etc. Usually, primers with 18-24 bp can lead to better yield. In order to prevent template damage, the denaturation time at 94°C should be reduced to 30 sec or less per cycle, and the time to rise temperature to 94°C before amplification should be less than 1 min. Moreover, setting the extension temperature at about 68°C and designing the extension time according to the rate of 1 kb/min can ensure effective amplification of long fragments.

Q: How to improve the amplification fidelity of PCR?

The error rate of PCR amplification can be reduced by using various DNA polymerases with high fidelity. Among all the Taq DNA polymerases found so far, Pfu enzyme has the lowest error rate and the highest fidelity (see attached table). In addition to enzyme selection, researchers can further reduce PCR mutation rate by optimizing reaction conditions, including optimizing buffer composition, concentration of thermostable polymerase and optimizing PCR cycle number.


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