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PREMIUM Bullseye Taq DNA Polymerases
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Store at -20°C. For in-vitro laboratory use only
General Description
Bullseye Taq DNA Polymerase is a thermostable recombinant DNA polymerase, which exhibits very high activity in primer extension and other molecular biology applications. The enzyme is isolated from Thermus aquaticus and has a molecular weight of approximately 94 kDa.
Bullseye Taq DNA Polymerase has both a 5'®3' DNA polymerase and a 5'®3' exonuclease activity. The enzyme lacks a 3'®5' exonuclease activity (no proofreading ability). Taq DNA Polymerase leaves an A¢ overhang, which makes the enzyme ideal for TA cloning.
The 10X Reaction Buffer provided does not contain Mg+2. 25 mM MgCl2 is supplied separately.
10X Mg++ Free Standard Buffer
100 mM Tris-HCl pH 8.3, 500 mM KCl.
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 and Dilution Buffer
Enzyme is supplied in 20 mM Tris-HCl pH 8.3, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5%NP40, 50% glycerol.
Quality Control
Each lot of Taq DNA Polymerase is tested for contaminating activities, with no trace of endonuclease activity, nicking activity, exonuclease activity or priming activity.
Suggested Protocol using Taq DNA Polymerase
This protocol serves as a guideline only. Optimal reaction conditions may vary and must be individually determined.
n In some applications, MgCl2 is needed for the best results. For this reason, 25 mM MgCl2 is included with the kit.
1. Thaw 10X Mg2+ Free Standard Buffer, dNTP mix, 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.
Table 1. Reaction components (master mix & template DNA)
Component |
Vol./reaction |
Final Conc. |
10X Mg2+ Free Buffer |
5 µl |
1X |
MgCl2, 25 mM |
1- 9 µl |
0.5 4.5 mM |
dNTP mix (12.5 mM of each) |
0.8 µl |
0.2 mM of each dNTP |
Primer A |
Variable |
0.11.0 µM |
Primer B |
Variable |
0.11.0 µM |
Taq DNA Polymerase |
Variable |
1-5 units |
Template DNA |
Variable |
Variable |
Distilled Water |
Variable |
- - - - |
TOTAL volume |
50 µl |
- - - - |
Table 2. MgCl2 concentration in a 50 mL reaction
Final MgCl2 conc. in reaction (mM) |
0.5 |
1.0 |
1.5 |
2.0 |
2.5 |
3.0 |
3.5 |
4.0 |
4.5 |
Volume of 25 mM MgCl2 per rxn (µl): |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
Table 2 provides the volume of 25 mM MgCl2 to add to the master mix if a certain MgCl2 concentration is required.
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 to the individual tubes containing the master mix.
5. Program the thermal cycler according to the manufacturers 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.
PNPCRREAG
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