Red-Taq DNA Polymerase 5 U/ul 10x Standard Buffer, Mg2+ free 10,000U

Description

Red-Taq DNA Polymerase

With 10X Mg²⁺ Free Standard Buffer

 

5 units/µL

 

 

Cat. No.	Units	10X Standard Buffer  (Mg2+ Free)	MgCl2 25 mM
AO202203	500	1.5 mL	1.5 mL
AO202204	1,000	2 x 1.5 mL	2 x 1.5 mL
AO202206	2,500	4 x 1.5 mL	4 x 1.5 mL
AO202207	5,000	3 x 5 mL	3 x 5 mL
AO202208	10,000	6 x 5 mL	6 x 5 mL

Store at -20°C.        For in-vitro laboratory use only

 

General Description

 

AS ONE Red Taq DNA Polymerase is a thermostable recombinant DNA polymerase, which exhibits very high activity in primer extension and other molecular biology applications. Taq contains a red dye which provides easy and quick identification of reactions to which enzyme was added and allows confirmation of complete mixing. The inert dye has no effect on downstream processes. Taq with Red Dye is added directly to the reaction mix and is used in the same manner as standard Taq DNA Polymerase.

 

AS ONE Red Taq DNA Polymerase has both a 5’®3′ DNA polymerase and a 5’®3′ exonuclease activity.  The enzyme lacks a 3’®5′ exonuclease activity. Taq DNA Polymerase leaves an A¢ overhang, which makes the enzyme ideal for TA cloning.

 

Key Features

• High performance thermostable DNA polymerase
• Red dye identifies tubes which contain enzyme and confirms complete mixing of reagents
• Leaves an A¢ overhang

 

Unit Definition

One unit is defined as the amount that incorporates 10 nmoles of dNTPs into acid-precipitable form in 30 minutes at 72°C under standard assay conditions.

 

Storage Buffer

Enzyme is supplied in 20 mM Tris-HCl pH 8.3, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, inert dye, 0.5 % Tween^Ò 20, 0.5% NP40, 50% glycerol.

 

10X Mg⁺² Free Standard Buffer

100 mM Tris-HCl pH 8.3, 500 mM KCl, 1% Triton X-100

 

Quality Control

Endonuclease, exonuclease and priming activities are not detected after 3 hours incubation of 1 mg of pUC19 plasmid DNA and 0.5 µg Eco RI digested lambda phage DNA at 72°C in the presence of 40 units of Red Taq DNA Polymerase.

 

Suggested Protocol using Red Taq Polymerase

 

This protocol serves as a guideline. Optimal reaction conditions such as incubation times, temperatures, and amount of template DNA may vary and must be individually determined.

 

1. Thaw 10X Mg⁺² Free Standard Buffer, dNTP mix, and primer solutions. It is important to mix the solutions completely before use to avoid localized concentrations of salts.

 

2. Prepare a master mix according to Table 1. The master mix typically contains all the components needed for extension except the template DNA.

 

The optimal MgCl₂ concentration should be determined empirically but, in most cases a concentration of 1.5 mM produces satisfactory results. Table 2 provides the volume of 25mM MgCl₂ to add to the master mix if a higher MgCl₂ concentration is required.

 

Table 1.  Reaction components (master mix & template DNA)

Component	Vol./reaction	Final Conc.
10X Mg Free Buffer	5 µL	1X
25 mM MgCl2	3 µL (1-8 µL)	1.5 mM                  (0.5–4 mM)
dNTP mix (12.5 mM of each)	0.8 µL	0.2 mM of each dNTP
Primer A	1 µL (0.5-5 µL)	0.2 µM                  (0.1–1.0 µM)
Primer B	1 µL (0.5-5 µL)	0.2 µM                  (0.1–1.0 µM)
Taq DNA Polymerase	0.6 µL              (0.2–1 µL)	3 units (1–5 units)
Template DNA	Variable	Genomic DNA 50 ng (10-500 ng) Plasmid DNA 0.5 ng (0.1-1 ng) Bacterial DNA 5 ng (1-10 ng)
Distilled Water	Variable	– – – –
TOTAL volume	50 µL	– – – –

 

Table 2.  MgCl₂ concentration

 

Final MgCl2 conc. in reaction (mM)	0.5	1.0	1.5	2.0	2.5	3.0	3.5	4.0
Additional volume of 25 mM MgCl2 per reaction (µL):	1	2	3	4	5	6	7	8

 

3. Mix the master mix thoroughly and dispense appropriate volumes into reaction tubes. Mix gently (e.g., by pipetting) the master mix up and down a few times.

 

4. Add template DNA (0.1–0.5 mg/reaction) to the individual tubes containing the master mix.

 

5. Program the thermal cycler according to the manufacturer’s instructions.

 

For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.

 

6. Place the tubes in the thermal cycler and start the reaction.

 

7. After primer extension, load 5-10 mL of a 50 mL reaction directly on an agarose gel for analysis.

 

Three- step PCR Program:

 

Cycles	Duration of Cycles	Temperature
1	2-5 minutesa	95°C
25-35	20-30 secondsb 20-40 secondsc 30 secondsd	95°C 50-65°C 72°C
1	5 minutese	72°C

 

 

 

 

 

 

 

 

^a  Initial denaturation step. Optional, but recommended for genomic DNA

^b Denaturation step: Heating the DNA template disrupts the hydrogen bonds yielding ssDNA.

^c Annealing step: Primers anneal to ssDNA template. Typically annealing temperature is 3-5°C below the Tm of the primers.

^d Extension/elongation step: R-Taq DNA polymerase synthesizes a new DNA strand complementary to the DNA template. Extension time depends on length of DNA fragment to be amplified. At optimum temperature the DNA polymerase will polymerize 1000 bases per minute.

^e Final elongation step: After the last PCR cycle to ensure that any remaining ssDNA is fully extended.