Note The system contains sufficient reagents suitable for 200 amplification reactions of 50 μl each.
PCR Mix (2X) is an optimized ready-to-use PCR mixture of Recombinant Taq DNA Polymerase, PCR Buffer, MgCl2 and dNTPs. PCR Mix (2X) contains all components for PCR, except DNA template and primers.
Numerous applications for which a high-quality thermostable DNA polymerase is required
One unit is defined as the amount of the enzyme required to catalyze the incorporation of 10 nM of dNTPs into an acid-insoluble form in 30 minutes at 70°C using hering sperm DNA as substrate.
0.25U/ul Taq DNA polymerase, 2X PCR buffer, 0.4mM dNTPs, 3.2 mM MgCl2, 0.02% bromophenol blue.
Store at -20°C
ü PCR Mix is an optimized ready-to-use PCR mixture of Recombinant Taq DNA Polymerase which is the enzyme of choice for most PCR applications and the half-life of enzyme is >40 minutes at 95°C.
ü The error rate of Taq DNA Polymerase in PCR is 2.2x10-5 errors per nt per cycle; the accuracy
(an inverse of the error rate) an average number of correct nucleotides incorporated before making an error, is 4.5x104.
ü PCR Mix accepts modified nucleotides (e.g. biotin-, digoxigenin-, fluorescent-labeled nucleotides) as substrates for the DNA synthesis.
Basic PCR Protocol
1. Add the following components to a sterile 50 μl microcentrifuge tube sitting on ice:
2. Mix contents of tube and overlay with 50 μl of mineral or silicone oil.
3. Cap tubes and centrifuge briefly to collect the contents to the bottom.
4. Incubate tubes in a thermal cycler at 94°C for 3 minutes to completely denature the template.
5. Perform 25-35 cycles of PCR amplification as follows:
6. Incubate for an additional 10 min at 72°C and maintain the reaction at 4°C. The samples can be stored at -20°C until use.
Notes on cycling conditions
ü Initial denaturation can be performed over an interval of 1-5 min at 95℃ depending on the GC content of template.
ü 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 1-2℃.
ü The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. 25-35 cycles are usually sufficient for the majority PCR reaction. Low amounts of starting template may require 40 cycles.
ü The time of the final extension step can be extended for amplicons that will be cloned into T/A vectors.
7. Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.