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Extension Time In Pcr In Oakland
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Oakland
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The third step in a PCR cycle is the extension step. The extension step, also referred to as the elongation step, is the PCR step in which Taq polymerase adds nucleotides to the annealed primer. The process of repeating the denaturation, annealing and extension steps of PCR is known as PCR cycling.
If the extension time is too short, there will be insufficient time for complete replication of the target. Generally, use an extension time of 1 min/kb. If the annealing time is too short, primers do not have enough time to bind to the template. Use an annealing time of at least 30 sec.
Many of the common problems with PCR and RT-PCR are identified during agarose gel electrophoresis of the reaction products. These include the absence of the expected amplification product, the presence of nonspecific products, excessive smearing, and the presence of a “primer dimer” band.
Final Extension A post-PCR final incubation step of 5–10 min at 72°C is often recommended to promote complete synthesis of all PCR products.
The extension time of PCR depends upon the synthesis rate of DNA polymerase and the length of target DNA. The typical extension time for Taq DNA Polymerase is 1 min/kb, whereas that of Pfu DNA polymerase is 2 min/kb.
Extension is achieved by using the loosened nucleotides of each base to grow the complementary DNA strand. The end result is two double-stranded products of DNA. The temperature that is used during the extension phase is dependent on the DNA polymerase that is used.
Increasing the final extension time improves full-length replication and yield of a 0.7-kb, GC-rich PCR fragment from human gDNA in these experiments. The smear under the desired band in 0 minute final extension suggests incomplete extension of the PCR amplicon by the DNA polymerase.
Extension Time Extensions are normally performed at 68°C. As a general rule, use extension times of one minute per 1000 base pairs (e.g. 3 minutes for a 3 kb product) For products less than 1 kb, use 45-60 seconds. Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions.
A standard QPCR reaction takes approximately 1 hour 45 minutes to complete. This includes an initial step of 15 minutes at 95 °C to activate the chemically modified hot-start Taq DNA polymerase, normally followed by 35–45 cycles of a 15-second denaturation at 95 °C and then 60 seconds annealing and extension at 60 °C.
Duration of extension will be dependent upon amplicon size (30 sec per 1 kb). The period of elongation depends upon the desired length of the amplicon and the enzyme used. Since qPCR amplicons are short, this is typically 5–30 sec.
