TaKaRa Titanium Taq

TaKaRa Titanium Taq is a family A enzyme from Unknown.

Titanium Taq is an antibody-based hot start DNA polymerase available from TaKaRa with the product number 639208. 

 

The following primer design recommendations are provided:

Primer design is the single largest variable in PCR applications and the single most important factor in determining the success or failure of PCR reactions. Always check your primer design before constructing or ordering primers. Titanium Taq SP DNA Polymerase (Cat. No. 639292) can be used in a wide variety of PCR applications, and the constraints on primer design will vary from one application to the next. In general, primers should have a Tm of ~70°C to achieve optimal results in a two-step cycling program with a 68°C combined annealing/extension step. Therefore, whenever possible, primers should be at least 22 nucleotides long (25–30-mers are preferred) and should have a GC content of 45–60%. Furthermore, the 3’ ends of each primer should not be complementary to each other and should have a low GC content.

 

Reaction setup is suggested as follows:

Volume           Reagent

  40 μl            PCR-Grade Water

  5 μl              10X Titanium Taq SP PCR Buffer

  1 μl              50X dNTP Mix (10 mM ea.)

  1 μl              5’ primer (10 μM)

  1 μl              3’ primer (10 μM)

  1 μl              50X Titanium Taq SP DNA Polymerase

  1 μl              DNA Template (100 ng/μl)

50 μl Total Volume

 

Cycling conditions are recommended as follows:

Target Size Cycle Parameters <5 kb:

• 95°C for 1 min

• 25–35 cycles ^A

        95°C for 30 sec^B

        68°C for 1–3 min^C

• 68°C for 3 min^D

Target Size Cycle Parameters >5 kb: Use Advantage 2 Kit

A Use 25 cycles for multiple-copy genes or medium-to-high abundance cDNAs and 30–35 cycles for single- or low-copy-number genes or rare cDNAs. For most applications, we prefer two-step cycles (denaturation at T1 followed by annealing and extension at T2) instead of three-step cycles (denaturation at T1 followed by annealing at T2 followed by extension at T3)—unless the Tm of the primers is <60–65°C and in certain special protocols.

B Use the shortest possible denaturation time. In some cases, better results may be obtained by modifying the denaturation step (15 sec, 94°C). Exposure of DNA to high temperatures causes some depurination of single-stranded DNA during denaturation, which eventually leads to strand scission. High temperature also leads to gradual loss of enzyme activity. Minimizing denaturation time is particularly important in experiments with very large templates where total cycling time can exceed 12 hr.

C Use the longest possible annealing/extension temperature. See Note A. Some researchers prefer to use an annealing/extension time equal to the expected target size (in kb) plus two minutes. We recommend using 1 min per kb of expected target.

D Optional: This final extension may reduce background in some cases. 

• Create new Polymerase Lot based on TaKaRa Titanium Taq
• Create new Formulation based on TaKaRa Titanium Taq

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