Platinum®TaqDNA Polymerase High Fidelity contains recombinant TaqDNA polymerase, Pyrococcus speciesGB-D polymerase, and Platinum®TaqAntibody. This enzyme allows amplification of simple and complex DNA templates over a large range of target sizes and provides 6X higher fidelity over Taq. Taq DNA Polymerase is the industry standard for routine PCR. Taq with Standard Taq Buffer ( NEB #M0273) is available in economical extra-large pack sizes. NEB provides high quality recombinant Taq at an exceptional value. Taq is available with different formats to accommodate a variety of PCR applications. TECHNICAL REFERENCE PROMEGA CORPORATION 2800 WOODS HOLLOW ROAD MADISON, WI 53711-5399 USA TELEPHONE 608-274-4330 promega.com ©2010 ALL RIGHTS RESERVED PART #GE639 Properties of Thermostable DNA Polymerases Taq DNA Polymerase is a thermostable DNA Polymerase isolated from an E. coli strain that carries the Taq DNA polymerase gene. Taq DNA polymerase is the most common polymerase used for PCR* reactions II. APPLICATIONS Taq DNA Polymerase can be used in most applications including the following: PCR. 3' A-tailing of blunt ends. TaqDNA Polymerase and the TaqPCR Core Kit are shipped on dry ice but retain full activity at room temperature (15-25°C) for 2 weeks. The Taq PCR Master Mix Kit is shipped on dry ice but retains full activity at room temperature (15-25°C) for 3 days. Taq DNA Polymerase, the Taq PCR Core Kit, and the Taq PCR Master Mix Kit, Platinum® Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks polymerase activity at ambient temperatures. Activity is restored after the denaturation step in PCR cycling at 94°C, thereby providing an automatic "hot start" for Taq DNA polymerase in PCR (1,2,3). Hot starts in PCR provide 1.5 µL 1.5 mM 10 mM dNTP mix 1 µL 0.2 mM each KB Extender (optional)* 1.5-4.5 µL 3-9% Platinum™TaqDNA Polymerase 0.2 µL 2 U/rxn *For targets >5 kb or with >65% GC sequences. Mix and then briefly centrifuge the components. 3 Add template DNA and primers Add your template DNA and primers to each tube for a final reaction volume of 50 μL. Taq DNA Polymerase is supplied with two buffers: Taq buffer with KCl and Taq buffer with (NH 4) 2 SO 4. K+ stabilizes primer annealing whereas NH 4 + has a destabilizing effect especially on weak hydrogen bonds between mismatched primer - template base pairs. Therefore for standard PCR with Taq DNA Polymerase and 0.2 mM dNTPs the recommended MgCl 2 TaKaRa Ex Taq (5 U/μl) 0.25 μl 10X Ex Taq Buffer (Mg2＋ plus) (20 mM) 5 μl dNTP Mixture (2.5 mM each) 4 μl Template < 500 ng Primer 1 0.2 - 1.0 μM (final conc.) Primer 2 0.2 - 1.0 μM (final conc.) Sterile purified water up to 50 μl Example of PCR conditions : When amplifying a 1 kb DNA fragment 98℃ 10 sec 55℃ 30 sec 72℃0.5% 1 min 1 μl 50X Titanium Taq SP DNA Polymerase 1 μl DNA Template (100 ng/μl) 50 μl Total Volume III. Recommended Cycling Conditions Use the following guidelines when setting up your initial experiments with Titanium Taq SP DNA Polymerase. These are general guidelines—the optimal cycling conditions may vary with different thermal cyclers and will Taq polymerase • DNA polymerase purified from Thermus aquaticus a bacterium living in hot springs • replicates DNA by incorporating dNTPs on 3' OH end on a primer hybridized to a DNA matrix • Mg2+ is a co-factor • Optimal temperature for activity 72 C • Purified in 1976 by Chien and colleagues Taq DNA Polymerase ensures highly specific PCR for a range