2×Fast Pfu PCR Master Mix

Composition

Storage

The storage period is more than 2 years at -20℃, and the validity period is 6 months when stored at 2~8℃.

Introduction

The 2×Fast Pfu PCR Master Mix is an optimized PCR master mix with a double concentration of PCR master mix with extremely high amplification efficiency and broad template adaptability for almost all PCR reactions. The product is easy to use, just take 2× Fast Pfu PCR Master Mix 0.5 times of the volume of the PCR system, add primers and template, and make up the volume with ddH₂O.

2×Fast Pfu PCR Master Mix contains fast Pfu DNA Polymerase, which has 52 times the fidelity of ordinary Taq DNA polymerase and 6 times that of Pfu DNA polymerase, and the amplification rate can reach 15 sec/kb.

Components of the PCR system

1.     Purity of template DNA: Many residual nucleic acid extraction reagents can affect the PCR reaction, including proteases, protein denaturants (e.g., SDS, guanidine salts), high concentrations of salts (KAc, NaAc, sodium caprylate, etc.), and high concentrations of EDTA. Do not use more than 1/10 of the PCR reaction for less pure templates (e.g., 5 μl of the sample for 50 μl PCR system). If the template DNA purity is too poor, the DNA Purification Kit (Cat. No.2101050) is recommended to purify and concentrate the template DNA, and the amount of template used after purification can be as much as 1/2 of the volume of the PCR system.

2.     Template DNA dosage: A very small amount of DNA can also be used as a PCR template, but to ensure the stability of the reaction, it is recommended to use more than 10⁴ copies of the target sequence as a template for the 50 μl system. Recommended dosage of template DNA:

Human Genomic DNA:                   0.05 µg~0.5 µg/50 µl PCR system

E. coli genomic DNA:                     10 ng~100 ng/50 µl PCR system

λ DNA：                                           0.5 ng~5 ng/50 µl PCR system

Plasmid DNA：                               0.1 ng ~ 10 ng/50 µl PCR system

If the amplification product is to be used as a template for reamplification, the amplification product should be diluted at least 1,000 to 10,000-fold before using it as a template, otherwise smeared bands or non-specific bands may occur.

3.     Primer concentration: Typically, each primer is prepared at a concentration of 10 μM (50×) and a working concentration of 0.2 μM. Too much primer may result in non-specific amplification, and too little primer may reduce amplification efficiency.

PCR parameter settings

1. Initial denaturation: generally, initial denaturation is 94℃, 1~5 min. Denaturation temperature is too high or too long, and the activity of the fast Pfu enzyme is lost.

2. Annealing: Annealing temperature is key in PCR, too high a temperature may reduce yield, and too low a temperature may produce primer-dimers or non-specific amplification. For the first attempt at PCR amplification, it is recommended to try a lower than 5 ℃ Tm (if the two primer Tm are different, refer to the lower Tm) as the annealing temperature. Generally, primer synthesis companies will provide the Tm of the synthesized primers, and the primer Tm can also be estimated according to this formula: Tm = 2℃×(A+T) + 4℃× (G+C). The optimal annealing temperature needs to be determined by gradient PCR.

3. Extension: The extension temperature is typically 72℃, and the length of extension time depends on the length of the DNA fragment of interest, and the required extension time is calculated at 15 sec/kb, too long a time may lead to a non-specific increase. After the end of the cycle, continue to extend for 5~10 min to obtain the complete double-stranded product.

4. Number of cycles: Generally, 25~35 cycles are used, and the number of cycles can be increased appropriately with a low copy template. Excessive number of cycles may increase non-specific amplification and decrease specific products.

Protocol

1.       Thaw 2× Fast Pfu PCR Master Mix, ddH₂O, template DNA, and primers at room temperature and place on ice.

2.       After thawing, the components were inverted up and down to mix evenly, and the PCR system was prepared according to the following table:

* Note: The above example is for the addition of components to a 50 μl reaction system, if additional volumes of the reaction are required, please increase or decrease the components proportionally.

3.       Flick the PCR reaction tube to mix well, and spin down the solution to the bottom of the tube.

4.       Example of PCR reaction cycle setup

※ The actual optimal annealing temperature shall prevail.

§ Calculated at 15 sec/kb.

For amplification of target fragments greater than 10 kb, a two-step amplification method is required:

※The temperature can be adjusted between 60~68℃, and the actual optimal temperature is subject to the actual optimal temperature, and the time is calculated as 15 sec/kb.

5.       Results: 5-10 μl of the amplified product was mixed with Loading Buffer and then detected by agarose gel electrophoresis.