This product is a 2X Long PCR reagent Mix, plus a separately supplied Long PCR DNA Polymerase. The mix contains dNTPs, Mg2+ and Buffer at optimal concentrations. This Optimzer format provides flexibility of optimizing Long DNA polymerase activity for each unique reaction and still the convenience that comes with master mixes. The Long Taq was shown to amplify long templates from λ phage genome of up to 20 kb. It is also a better choice for amplifying complex targets, such as GC-rich targets.
Long PCR Taq DNA Polymerase,a combination of two thermostable DNA polymerases, Taq and Pfu, is a special formulation designed for amplifying large fragment. This specially formulated Long PCR Taq was shown to amplify long templates from λ phage genome of up to 20 kb. It is also a better choice for amplifying complex template, such as GC-rich template.
Long PCR Taq is suitable as a direct replacement for ordinary Taq Polymerase in most applications. Using Long PCR Taq in your PCR reactions results in 3´-dA overhangs PCR products, which can be used in TA clone
5 x 1 ml is supplied.
Applications
• PCR amplification of DNA fragments any sizes around 5 kb
• DNA labeling
• DNA sequencing
• PCR for cloning
Features
• High fidelity: three times fidelity of Taq DNA Polymerase.
• Longer fragment: amplify long templates as long as 40kb.
• Amplification of complex template (GC rich or repetitive sequence).
• Generates 3'-dA and blunt end PCR products.
Note:
- 10 x Long PCR Buffer is classical Long PCR Taq DNA Polymerase buffer, is good for long template especially above 10kb.
- 10 x Long PCR Buffer Ⅱ is an alternative long PCR buffer . It is for better fidelity but may not be robust for longer templates above 10 kb.
Notes on cycling conditions
- Initial denaturation can be performed over an interval of 1~5 min at 95℃ depending on the GC content of template.
-Denaturation for 30 sec to 2 min at 94~95℃ is sufficient. If the amplified DNA has a very high GC content, denaturation time may be increased up to 4 min.
-Optimal annealing temperature is 5℃ lower than the melting temperature of primer-temperature DNA duplex. If nonspecific PCR products are obtained optimization of annealing temperature can be performed by increasing temperature stepwise by 2℃.
-The number of PCR cycles depends on the amount of emplate DNA in the reaction mix and on the expected yield of the PCR products, 25-35 cycles are usually sufficient
for the majority PCR reaction. Low amounts of starting template may require 40 cycles.
-The time of the final extensi, on step can be extended for amplicons that will be cloned into T/A vectors.