Extension Time In Pcr In Fulton
Description
Form popularity
FAQ
If the annealing temperature is too low, non-specific products can be generated, leading to smeared bands on the gel. If the extension time is too long, secondary products can be formed, which can contribute to smearing.
A 1 min extension is typically sufficient to synthesize PCR fragments up to 2 kilobases (kb). To amplify larger fragments, the elongation step is extended at a rate of 1 min per kb. During the first extension, the template will not be length limiting and so templates will be synthesized that exceed the amplicon length.
Primer length Longer primers are less efficient during the annealing step, resulting in a lower amount of PCR product.
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.
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.
If the annealing temperature is too low, non-specific products can be generated, leading to smeared bands on the gel. If the extension time is too long, secondary products can be formed, which can contribute to smearing.
Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each ...
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. If the annealing temperature is too high, primers are unable to bind to the template.
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.
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.
