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Extension Time In Pcr In Tarrant
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State:
Multi-State
County:
Tarrant
Control #:
US-0018LTR
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Word;
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FAQ
Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low. 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.
During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times (typically 25-35 cycles), and because each new strand can also serve as a template for the primers, the region of interest is amplified exponentially.
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.
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.
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.
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.
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.
One-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube. However, two-step RT-PCR entails two separate reactions, beginning with first-strand cDNA synthesis, followed by amplification of a portion of the resulting cDNA by PCR in a separate tube.
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.
