Extension Time In Pcr In Harris
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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.
The final stage is the extension step (20 sec to 1 min at 72 °C), which is performed so that the DNA polymerase extends the primer sequences from the 3' of each primer to the end of the amplicon. A 1 min extension is typically sufficient to synthesize PCR fragments up to 2 kilobases (kb).
Polymerase chain reaction (PCR) is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment.
If the extension time is too long, secondary products can be formed, which can contribute to smearing. The quality of the template DNA can also affect the appearance of the PCR product on the gel. Degraded DNA can lead to the formation of shorter and larger fragments, which can contribute to smearing.
The final stage is the extension step (20 sec to 1 min at 72 °C), which is performed so that the DNA polymerase extends the primer sequences from the 3' of each primer to the end of the amplicon. A 1 min extension is typically sufficient to synthesize PCR fragments up to 2 kilobases (kb).
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.
Protocol STEPTEMPTIME Initial Denaturation 98°C 30 seconds 25–35 Cycles 98°C 5–10 seconds 72°C 15–30 seconds/kb Final Extension 72°C 2 minutes1 more row •
Primer length Longer primers are less efficient during the annealing step, resulting in a lower amount of PCR product.
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.
