G&P HiFi™ DNA polymerase, with 5xHD reaction buffer

Product ID: G&P HiFi DNA Pol (SKU#: P01302)

Price:

$95.00

Description

G&P HiFi™ is a Thermococcus proofreading DNA polymerase engineered for superior fidelity and performance. The engineered enzyme contains amino acid modifications conferring dramatic improvements through screening a large library of enzyme variants. G&P HiFi™ is the most accurate thermostable DNA polymerase, featuring an error rate over 100-fold lower than Taq, making it a superior choice for applications requiring high fidelity. G&P HiFi™ DNA polymerase is supplied with two proprietary buffers for optimal performance and compatibility with downstream applications. Combined with the proprietary reaction buffers that enhance specific primer annealing and accurate DNA replication, successful amplification of a wide range of amplicon types and sizes (up to 15 kb) can be achieved using a single enzyme. G&P HiFi™ requires only 10-20 sec per kb extension time per cycle, making it possible to perform PCR with extreme speed. It also produces higher yields with lower enzyme amounts than other common high fidelity DNA polymerases. The PCR products amplified by G&P HiFi™ are blunt-ended, suited for various molecular applications, including gene cloning, sequencing, mutagenesis and assembly.

Product Details

Reagents Supplied:

The enzyme (SKU#E01301) is supplied at 2 U/ml in the enzyme storage buffer (20 mM Tris-HCl pH7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.05% Tween-20, 0.05% NP-40 and 50% glycerol). The DNA polymerase is provided with a 5X HD (High-fidelity Detergent-free, SKU#B01302) reaction buffer containing 7.5 mM MgSO4 (for a final working concentration at 1.5 mM). The HD buffer is uniquely formulated for optimal performance and compatibility with downstream applications that are sensitive to detergents. It is recommended for high fidelity PCR, enabling consistently high success rate. Additional materials and reagents, such as the DNA template, primers, PCR-grade dNTPs and water, are required but not included in the product package.

Quality Control Assays:

G&P HiFi™ DNA polymerase is extensively purified and free of any contaminating nuclease activity. It meets strict quality requirements. It is tested by PCR of multiple DNA templates with sizes ranging from 0.4 to 12.5 kb: 30 cycles of PCR in 50 μl reactions containing 0.1 pg – 1 ng of DNA template with 1 U of G&P HiFi™ DNA polymerase, 200 μM dNTPs, and 0.3 μM primers in 1x HD reaction buffer resulting in the expected 0.4, 1.2, 2.5, 3.4, 5.0, 7.5 and 12.5 kb amplified DNA products (see G&P HiFi™ PCR System for more details and data examples).

General Reaction Conditions:

1XHD reaction buffer with 0.1 pg–10 ng of low complexity DNA templates such as plasmid DNA or 50–500 ng of high complexity DNA templates such as genomic DNA, 0.2–0.5 μM primers, 200 μM dNTPs, 0.5–2 U of G&P HiFi™ in a total volume of 50 μl. Typically 1 U of DNA polymerase per 50 μl reaction will give satisfactory PCR, but optimal amounts could range from 0.5–2 U depending on amplicon length or GC content. Do not use >2 U per 50 μl reaction. Remember to include a negative control (e.g., everything but lacking DNA template) and a positive control (e.g., using a DNA template known to amplify with the primers) if desired. Amplification of DNA templates with extremely high GC content (e.g., >70%), complex secondary structure, low template amount (e.g., <0.1 pg), or long amplicons (e.g., >15 kb) may require further optimization (see "Standard Protocol" for more details).

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Notice to purchaser: G&P HiFi™ PCR products (patent pending) are developed and sold exclusively for research purposes. Neither the products, nor any individual components, have been tested for use in humans or animals. Certain applications of this product are covered by patents issued to parties other than G&P Biosciences and may be applicable in certain countries. Purchase of this product does not include a license to perform any such applications. The purchase of this product includes a limited, non-transferable immunity for using only this amount of product for the purchaser’s own internal research. Users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used.

Storage

The product is shipped at 4°C. Upon receipt, centrifuge the product briefly before opening the vial. It is recommended to store small aliquots at the temperature below –20°C for long-term storage and the enzyme is stable for 6 months.

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Protocol

In most cases, the standard protocol described below will ensure successful PCR using G&P HiFi™. Remember to include a negative control (e.g., everything but lacking DNA template) and a positive control (e.g., using a DNA template known to amplify with the primers), if desired. Amplification of DNA templates with extremely high GC content (e.g., >70%), complex secondary structure, low template amount (e.g., <0.1 pg), or long amplicons (e.g., >15 kb) may require further optimization (see more information in "Troubleshooting").

General Conditions (final concentration):

1x HD PCR reaction buffer
0.1 pg–10 ng of low complexity DNA templates (e.g., plasmid DNA) or 50–500 ng of high complexity DNA templates (e.g., genomic DNA)
0.2–0.5 μM of 5'-primer
0.2–0.5 μM of 3'-primer
200 μM dNTPs
0.5–2 U of G&P HiFi™ DNA polymerase

Add PCR-grade water to a total volume of 50 μl per reaction. Typically 1 U of DNA polymerase per 50 μl reaction will give satisfactory PCR, but optimal amounts could range from 0.5–2 U depending on amplicon length or complexity. In general do not use >2 U per 50 μl reaction.

Reaction Setup: IMPORTANT NOTE – Please read before starting.

We recommend spinning all vials briefly prior to use and assembling a 2x enzyme master mix on ice prior to use (i.e., pre-diluting G&P HiFi™ DNA polymerase into 2x HD buffer and 400 μM dNTPs for the appropriate number of DNA samples to be amplified). This will minimize the 3’-5’ exonuclease activity of DNA polymerase and reduce pipetting steps or errors. We observe that pre-diluting G&P HiFi™ into the reaction buffer and priming it with dNTPs reduce its 3’-5’ exonuclease activity to an undetectable level, thereby preventing primer degradation. The 2x enzyme mix can then be added at 1:1 volume ratio to each DNA and primers mix, which can be prepared separately in a PCR tube. The reaction mixtures can then be transferred to a thermocycler without preheating to the initial denaturation temperature (i.e., a “hot start” condition is usually unnecessary).

Quick Protocol:

Step 1. For each 50 μl PCR reaction, assembly 25 μl of following 2x enzyme master mix on ice prior to use:

10.0 μl 5x HD reaction buffer (final concentration at 1x)
1.0 μl dNTP mixture (10 mM each, final concentration at 0.2 mM)
0.5 μl G&P HiFi™ PCR DNA polymerase (2 U/μl, 1 U per reaction)
13.5 μl PCR-grade water to a total volume of 25 μl per reaction

Note: Calculate and prepare the appropriate number of 25-μl aliquot of 2X enzyme master mix (usually at least 10% more than needed) adequate for all DNA samples to be amplified.

Step 2. For each DNA template to be amplified, prepare 25 μl of DNA and primers mix in a 0.2 ml PCR tube:

x μl DNA template (typically 1 ng of plasmid DNA; 200 ng of genomic DNA)
1.5 μl 5’ primer (10 μM stock, final concentration 0.3 μM)
1.5 μl 3’ primer (10 μM stock, final concentration 0.3 μM)
22-x μl PCR-grade water to a total volume of 25 μl per DNA sample to be amplified

Step 3. Aliquot 25 μl of 2x enzyme master mix into each tube of DNA and primers mix. Gently mix the reaction by pipetting up and down a few times. Spin briefly to collect all liquid to the bottom, if necessary. Overlay the samples with mineral oil if using a thermocycler without a heated lid. Otherwise place the reaction tubes in the thermolcycler and begin thermocycling as follows:

Cycling Step	Temperature	Time	Number of Cycles
Initial Denaturation	98°C	1 minute^a	1
Denaturation Annealing Extension	98°C 45-72°C (3-5°C below T_m^b) 72°C	5 seconds 15 seconds 10-20 seconds per kb^d	20-40^c
Final Extension	72°C	5 minutes	1
Hold	4°C

^aIn general use 1 minute initial denaturation at 98°C as a starting point. This can be extended to 1—3 minutes for difficult DNA templates such as genomic DNA and GC-rich template. Subsequent denaturation should be performed for 5–10 seconds at 98°C.

^bIn general use annealing temperature at least 3°C below the T_m (melting temperature) of the primers as a starting point. Optimize in 2°C increments if necessary.

^cIn general use 30 cycles as a starting point. Optimize in 2-3 cycles increments if necessary. In general do not exceed 40 cycles.

^dIn general use 15 seconds per kb for each round of extension as a starting point. Optimize in 5 seconds increments if necessary. Do not exceed 1 minute per kb.

Step 4.

Analyze PCR reactions by agarose gel electrophoresis and optimize reaction conditions, if necessary.

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

Guideline

Template DNA Quantity & Quality:

For optimal fidelity and specificity, use the lowest amount of template DNA possible. Amplification from low complexity DNA templates, such as plasmid DNA usually requires little optimization (1 ng template per 50 μl reaction is recommended). Amplification of low target copy number genes from high complexity templates such as genomic DNA is more challenging (200 ng or even more DNA may be required). High quality template DNA is essential for any high-fidelity PCR. Degraded, damaged or sheared template DNA is problematic. Always dilute and store DNA in TE pH8.0—8.5 buffer and minimize freeze-thaw cycles.

Oligonucleotide Primers:

Oligonucleotide primers are generally 20–50 nucleotides in length and ideally have a GC content of 40–60% (GC contents greater than 60% may require higher denaturation temperatures and/or longer denaturation times). Primer pairs should exhibit similar melting temperatures (T_m). The final primer concentration may be 0.2—0.5 μM, while 0.3 μM is recommended for high fidelity PCR using G&P HiFi™ DNA polyermase.

Mg²⁺ and dNTPs Concentration:

Mg²⁺ concentration is critical to achieve optimal performance with G&P HiFi DNA Polymerase. Excessive Mg²⁺ can prevent full denaturation of DNA and cause non-specific binding of primers. Conversely, insufficient amounts of Mg²⁺ can lead to low enzyme activity or product yield. The optimal Mg²⁺ concentration for G&P HiFi™ in 1X HS buffer has been optimized to 1.5 mM. Only high quality dNTPs should be used. Use of dUTP or analogs is not recommended. There is no advantage to increasing dNTP amounts. For optimal results, use 200 μM of dNTPs.

PCR Additives:

Amplification of difficult templates, such as those with high GC content (e.g., >70%) or secondary structure, may be improved by the presence of additives. A final concentration of 3—5% DMSO is recommended. It is important to note that if a high concentration of DMSO is used, the annealing temperature must be lowered as DMSO decreases primers’ T_m (e.g., 10% DMSO require lowering the annealing temperature by 6.0°C). Other PCR additives such as glycerol, betaine, ethylene glycol, and propanediol are compatible with G&P HiFi™ DNA polymerase for GC-rich amplicons.

Documentation