Hot Start Taq DNA Polymerase

Hot Start Taq DNA Polymerase Instructions

Composition

Storage

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

Introduction

Hot Start Taq DNA Polymerase is an enzyme solution of monoclonal antibodies against Taq enzyme mixed with Taq enzyme in 1:1 activity unit. Taq enzyme antibody has a very high affinity with Taq enzyme and can block Taq enzyme activity before denaturation at high temperature, so it can effectively inhibit primer dimerization and non-specific amplification, greatly improving the accuracy of PCR reaction.

Hot Start Taq DNA Polymerase can initiate the activity of Taq enzyme without special high-temperature treatment. Hot Start Taq DNA polymerase is suitable for a variety of Taq enzyme-based hot start PCR and qPCR reactions. The PCR product has A 3 'end and can be cloned directly using TA vector.

Unit Definition

The amount of enzyme required to incorporate 10 nm dNTP into an acid-insoluble precipitate at 74℃ for 30 min is defined as one unit of activity.

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

SDS-PAGE detects purity greater than 99%. No exogenous nuclease activity was detected, and no host DNA was detected by PCR, which can effectively amplify single copy genes in 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: Typically, each primer is formulated at 10 µM and the working concentration is 0.2 µM. Excessive primers may result in non-specific amplification, while too few primers may reduce amplification efficiency.

PCR Parameter Settings

1.        Pre-denaturation: the general pre-denaturation is 94℃, 1~5 min. Denaturation temperature is too high, or the denaturation time is too long will lose the activity of Taq enzyme.

2.        Annealing: Annealing temperature is key for PCR, too high temperature may reduce yield, too low temperature may produce primer dimer or non-specific amplification. The first attempt at PCR amplification is recommended to try below Tm 5℃(if the two primers Tm are different, refer to the lower Tm) as the annealing temperature. Generally, primer synthesis companies will provide the Tm of the synthesized primer, but 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 usually 72℃, and the length of elongation depends on the length of the destination DNA fragment. The elongation time required is calculated at 1 kb/min. Too long a time may lead to an increase in non-specificity. After the end of the cycle, continue to extend for 5 to 10 min to obtain a complete double-stranded product.

4.        Number of cycles: Generally, 25 to 35 cycles are used, and the low copy template can appropriately increase the number of cycles. But too many 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 thoroughly mixed before use, otherwise it will affect the PCR effect.

ž   The above examples are the components added to the 50 µl reaction system. If other volumes of reaction system are required, please increase or decrease the components in proportion.

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 setting.

94°C 3 min

94°C 30 sec

※55℃ 30 sec             30 Cycles

§ 72℃ 1 min

72°C 5 min

※The actual optimal annealing temperature shall prevail.

§ Calculated at 1 kb/min.

5.       Result detection: 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: