Taq DNA Polymerase (Mg 2+ Plus Buffer)
Catalog: MZ20600
Size: 1,000 U / 5,000 U / 10,000 U
Concentration: 5,000 U/ml
Product Components
| Component | Catalog Number |
|---|---|
| Taq DNA Polymerase(5,000 U/ml) | MZ20310 |
| 10X PCR Reaction Buffer, Mg²⁺ | MZ20101 |
Product Description
Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5’→3’ polymerase activity and a 5’ flap endonuclease activity. Taq DNA Polymerase was isolated from a recombinant source and offers robust and reliable reactions. It tolerates a wide range of templates and incorporates dUTP, dITP, and fluorescently-labeled nucleotides.
Product Source:
An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1.
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTPs into acid-insoluble material in 30 minutes at 75°C.
Reaction Conditions:
1X PCR Reaction Buffer, Mg²⁺ plus
1X PCR Reaction Buffer, Mg²⁺ Composition:
20 mM Tris-HCl
10 mM (NH₄)₂SO₄
10 mM KCl
2 mM MgSO₄
0.1% Triton X-100
pH 8.8 @ 25°C
Storage Temperature
-20°C
Storage Conditions
10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.5% Tween20, 0.5% NP-40, 50% Glycerol, pH 7.4 @ 25°C
Molecular Weight
Theoretical 94,000 daltons
Heat Inactivation
No
5'-3' Exonuclease
Yes
3'-5' Exonuclease
No
Strand Displacement
+
Resulting Ends
Single-base 3´ Overhangs
Error Rate
~ 285×10-6 bases
Instructions
~ 285×10-6 bases
Reaction Setup
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
Take 25/50 μL system as an example
| Composition | 25 μL | 50 μL | Final Conc. |
|---|---|---|---|
| ddH₂O | to 25 μL | to 50 μL | — |
| 10× PCR Buffer | 2.5 μL | 5 μL | 1× (Final 2 mM Mg²⁺) |
| 10 mM dNTP | 0.5 μL | 1 μL | 200 μM |
| Primer F (10 μM) | 0.5 μL | 1 μL | 0.2 μM (0.05 ~ 1 μM) |
| Primer R (10 μM) | 0.5 μL | 1 μL | 0.2 μM (0.05 ~ 1 μM) |
| Template DNA | Variable | Variable | <1 μg / 50 μL |
| Taq DNA Polymerase | 0.125 μL | 0.25 μL | 1.25 U / 50 μL (0.25–2.5 U / 50 μL) |
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Incubation in a thermocycler as the below program:
| Temperature | Time | Cycles |
|---|---|---|
| 95°C | 30 s | 1 |
| 95°C | 15-30 s | 30 |
| 45–68°C | 15–60 s | 30 |
| 68°C | 1 kb/min | 30 |
| 68°C | 5 min | 1 |
| 4–10°C | ∞ | 1 |
General Guidelines:
Template: Use of high-quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 μl reaction are as follows:
Genomic DNA: 1 ng–1 µg
Plasmid or viral DNA: 1 pg–1 ng
Primers: Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The final concentration of each primer in a PCR may be 0.05–1 μM, typically 0.1–0.5 μM.
Mg²⁺ and additives: Mg²⁺ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. Amplification of some difficult targets, like GC-rich sequences, may be improved with additives such as DMSO or formamide.
Deoxynucleotides: The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.
Taq DNA Polymerase Concentration: We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications.
Denaturation: An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minutes incubation at 95°C is recommended to lyse cells. During thermocycling, a 15–30 second denaturation at 95°C is recommended.
Annealing: The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.
Extension: The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.
Cycle number: Generally, 25–35 cycles yield sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.
2-step PCR: When primers with annealing temperatures above 68°C are used, a 2-step thermocycling protocol is possible.
Thermocycling Conditions for a Routine 2-Step PCR:
| Temperature | Time | Cycles |
|---|---|---|
| 95°C | 30 s | 1 |
| 95°C | 15–30 s | 30 |
| 65–68°C | 15–60 s | 30 |
| 68°C | 1 kb/min | 30 |
| 65–68°C | 5 min | 1 |
| 4–10°C | ∞ | 1 |
PCR product: The PCR products generated using Taq DNA Polymerase contain dA overhangs at the 3´ end; therefore, the PCR products can be ligated to dT/dU-overhang vectors.