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Bullseye PREMIUM PR DNA Polymerase

• Provides higher fidelity than standard Taq DNA Polymerase
• Produces blunt-ended fragments
• Processes <3 kb with extremely high fidelity

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

Bullseye PREMIUM PR DNA Polymerase is a thermostable enzyme with proofreading ability, which can be used in primer extension reactions and other molecular biology applications. PR Polymerase exhibits both 5'-3' DNA polymerase activity and 3'-5' proofreading exonuclease activity. It is recommended for applications, which require extremely high fidelity or blunt ending.

Optimal reaction conditions are achieved by using the 10x Ammonium buffer containing MgCl2 provided with the enzyme. 25 mM MgCl2 is also included separately, in case a higher MgCl2 concentration is required for a specific reaction.

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.

10X Ammonium Reaction Buffer
Tris-HCl pH 8.5, (NH4)2SO4, 1% Tween20,
15mM MgCl2

PR Storage Buffer
50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol, 0.1% NP40, 0.1% 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 PR DNA Polymerase.

Suggested Protocol using PR DNA Polymerase
 
This protocol serves as a guideline. Optimal reaction conditions 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, will produce satisfactory results. Table 2 provides the volume of 25 mM MgCl2 to add to the master mix if a higher MgCl2 concentration is required.

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

Table 2. MgCl2 concentration in a 50 uL reaction

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. PR is a proofreading enzyme and requires an extension time of 1-2 min/kb. For maximum yield and specificity, temperatures and cycling times should be optimized for each new template target or primer pair.