G&P HiFi™ PCR ready 2x master mix

Product ID: G&P HiFi PCR 2xMM (SKU#: M01303)

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™ 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. G&P HiFi™ PCR ready 2x master mix (SKU#M01303) is a convenient pre-mixed, ready-to-use 2x cocktail containing all ingredients for high fidelity PCR except the DNA template and primers. To start a PCR, only template DNA and primers need to be added by the user. The master mix is optimized for convenience, scalable to various reaction volumes using a simplified workflow for high throughput needs. The master mix also minimizes the number of pipetting steps (or errors) and saves valuable laboratory time.

Product Details

Reagents Supplied:

G&P HiFi™ PCR Ready 2x master mix is a convenient ready-to-use 2x cocktail containing G&P HiFi™ DNA polymerase, dNTPs, and optimized reaction buffer including MgSO4 and stabilizer in a formulation for easy and quick reaction setup. It contains all components for high fidelity PCR except DNA template and primers, which need to be simply added by the user. G&P HiFi™ 2x master mix offers robust and consistent performance for amplification of DNA up to 15 kb in length. 25 μl of G&P HiFi™ 2x master mix is required per 50-μl PCR reaction.

Quality Control Assays:

G&P HiFi™ PCR ready 2x master mix meets all quality requirements. It is tested by PCR of multiple DNA templates with amplicon lengths ranging from 0.4 to 12.5 kb: 30 cycles of PCR in 50 μl reactions containing 10 pg–1 ng of DNA templates and 0.3 μM of primers with 25 μl of G&P HiFi™ PCR ready 2x master mix result in the expected 0.4, 1.2, 2.5, 3.4, 5.0, 7.5 and 12.5 kb products (see G&P HiFi™ PCR System for more details and data examples).

General Reaction Conditions:

G&P HiFi™ PCR ready master mix is supplied at 2x concentration to allow approximately 50% of the final reaction volume to be used for the addition of primers and template: 0.1 pg–10 ng low complexity DNA templates (e.g. plasmid, phage, or viral DNA) or 50–500 ng high complexity DNA templates (e.g. genomic DNA) and 0.2–0.5 μM primers (0.3 μM is recommended) with final 1x G&P HiFi™ Master Mix in a total volume of 50 μl.

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™ PCR ready 2x master mix. 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:

Reaction Setup: IMPORTANT NOTE – Please read before starting.

For daily use, we recommend keeping small aliquots of G&P HiFi™ 2x master mix at -20 °C. Once thawed, mix and spin the vial briefly prior to use. To minimize nonspecific amplification and achieves more accurate PCR, we recommend using the “Cool Start Method”, i.e., keep all reagents on ice until use; prepare the reaction mixture on ice; set a thermal cycler ready to start with the designated program; transfer the PCR reactions to the thermal cycler and start thermal cycling immediately. For each DNA template to be amplified, prepare a DNA and primers mix in a PCR tube as described below in “Quick Protocol”. Then dispense equal volume of G&P HiFi™ PCR Ready 2x master mix into each tube containing DNA and primers in a total volume of 50 μl. The reactions can subsequently be transferred to the thermal cycler without preheating to the initial denaturation temperature (“hot start” condition is normally not required).

Quick Protocol:

Step 1. For each DNA template to be amplified, prepare 25 μl of template DNA and primers mix in a PCR tube with:

x μl DNA template (1 ng for plasmid DNA; 200 ng for genomic DNA)
1.5 μl 5’ primer (10 μM stock, for a final concentration 0.3 μM)
1.5 μl 3’ primer (10 μM stock, for a 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 2. Dispense 25 μl G&P HiFi™ PCR ready 2x master mix into each PCR tube containing the DNA template and primers. 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 thermocycler and begin thermal cycling 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 3.

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