Taq DNA Polymerase

Taq DNA Polymerase Instructions

Composition

Storage

It can be stored for up to two years at -20℃.

Introduction

Taq DNA Polymerase is a thermally stable protein, isolated and purified from a recombinant E. coli strain containing the Thermus aquaticus DNA Polymerase gene with a molecular weight of approximately 90 KD. It has 5'→3' polymerase activity and double-strand-specific 5'→3' exonuclease activity, and no 3'→5' exonuclease activity. The Taq DNA Polymerase PCR product is a 3' single A-sticky end that can be attached directly to the TA vector.

Unit Definition

The amount of enzyme required to incorporate 10 nm dNTPs into the acid-insoluble precipitate in 30 min at 74℃ was defined as one activity unit.

Activity assay conditions: 50 mM Tris-HCl (pH 9.0, 25℃), 50 mM NaCl, 5 mM MgCl₂, 0.2 mM each dNTPs (including [3H]-dTTP), 200 μg/ml activated calf thymus DNA and 0.1 mg/ml BSA.

Quality Control

The purity of the SDS-PAGE assay is greater than 99%. No foreign nuclease activity was detected, and no host DNA residue was detected by PCR method, which could effectively amplify single-copy genes in the human genome.

Components of PCR System

1.        Purity of template DNA: Many residual nucleic acid extraction reagents can affect the PCR, including proteases, protein denaturants (e.g. SDS, guanidinium salts), high concentrations of salts (KAc, NaAc, sodium caprylate, etc.) and high concentrations of EDTA. Do not use more than 1/10th of the PCR reaction system for poorly purified templates (e.g., templates obtained by boiling) (e.g., the volume of template added to a 50 µl reaction system should not exceed 5 µl). If the purity of the template DNA is too poor, the template DNA can be purified and concentrated by DNA Purification Kit (Cat. No. 2101050), and the amount of purified template used can be up to 1/2 of the volume of the PCR reaction system.

2.        Amount of template DNA: Trace amounts of DNA can also be used as PCR templates, but in order 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 reaction system. Recommended amount of template DNA:

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

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

λDNA:                                 0.5 ng ~ 5 ng / 50 µl PCR reaction system

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

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

3.        Primer concentration: Generally, each primer is formulated at 10 µM and the working concentration is 0.2 µM. Too much primer may result in non-specific amplification, and too little primer may reduce amplification efficiency.

PCR Parameter Settings

1.        Pre-denaturation: The general pre-denaturation is 94℃, 1~5 min. Denaturation temperatures that are too high or too long can result in loss of Taq enzyme activity.

2.        Annealing: Annealing temperature is key for PCR, as too high a temperature may reduce yield, and too low a temperature may result in 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.        Elongation: The elongation temperature is typically 72℃, and the length of elongation time depends on the length of the DNA fragment of interest, and the desired elongation time is calculated at 1 kb/min, which may result in 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. However, excessive number of cycles may increase non-specific amplification without increasing specific products.

Protocol

1.       Thaw 10×PCR Buffer (Mg²⁺), dNTPs, ddH₂O, template DNA, and primers at room temperature and place on ice.

2.       Invert the thawed components to mix evenly, and add each component in turn as shown in the following table to prepare a PCR reaction system:

Note:

ž   10×PCR Buffer (Mg²⁺) must be well mixed before use, otherwise the PCR effect will be affected.

ž   The above example is the addition of components for a 50 μl reaction, if additional volumes of the reaction are required, increase or decrease the components proportionally.

3.       Flick the PCR reaction tube with your finger to mix well, and centrifuge at low speed for a few seconds to settle the solution to the bottom of the tube.

4.       Example of PCR reaction cycle setup.

94℃ 3 min

94℃ 30 sec

※55℃ 30 sec           30 Cycles

§ 72℃ 1 min

72℃ 5 min

※The actual optimal annealing temperature shall prevail.

§ Calculated at 1 kb/min.

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

Relationship between agarose concentration and the optimal resolution range of linear DNA: