R-Taq DNA Polymerase 5 units/µL
| Cat. No. | Units | 10X Ammonium Buffer
(MgCl2 15mM) | MgCl2
25 mM |
| BE200303 | 500 | 1.5 mL | 1.5 mL |
| BE200304 | 1,000 | 2x 1.5 mL | 2x 1.5 mL |
| BE200306 | 2,500 | 4x 1.5 mL | 4x 1.5 mL |
Store at -20°C. For in-vitro laboratory use only
General Description
Bullseye R-Taq DNA Polymerase is a thermostable recombinant DNA polymerase, which exhibits very high activity in primer extension and other molecular biology applications. R-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. R-Taq is added directly to the reaction mix and is used in the same manner as standard Taq DNA Polymerase.
Bullseye R-Taq DNA Polymerase has both a 5'®3' DNA polymerase and a 5'®3' exonuclease activity. The enzyme lacks a 3'®5' exonuclease activity. R-Taq DNA Polymerase leaves an A-overhang, which makes the enzyme ideal for TA cloning.
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 % Tweenà 20, 0.5% NP40, 50% glycerol.
Component | Vol./reaction | Final Conc. |
10X Ammonium Buffer | 5 µL | 1X |
dNTP mix (12.5 mM each) | 0.8 µL | 0.2 mM each dNTP |
Primer A | Variable | 0.1-0.5 µM |
Primer B | Variable | 0.1-0.5 µM |
R-Taq DNA Pol | 1 mL | 5 units/reaction |
Distilled Water | Variable | - - - - |
Template DNA | Variable | 0.1-0.5 µg/reaction |
TOTAL volume | 50 µL | - - - - |
Table 2. MgCl2 concentration
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.
Tween 20 is a registered trademark of ICI Americas, Inc.
Final MgCl2 conc. in reaction (mM) | 1.5 | 2.0 | 2.5 | 3.0 | 3.5 | 4.0 | 4.5 |
Additional volume of 25 mM MgCl2 per reaction (µL): | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
10X Ammonium Reaction Buffer
Tris-HCl pH 8.5, (NH4)2S04, 15 mM MgCl2, 1% Tween 20
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 EcoR I digested lambda phage DNA at 72°C in the presence of 40 units of R-Taq DNA Polymerase.
Suggested Protocol using R-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 Ammonium 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, as provided in the 1X Ammonium Buffer, 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)
