![]() Q3: Amplification specificity is poor, or there are non-specific products. Try the touchdown PCR program increase the extension time and the number of cycles. If the template has been used up, use serial dilutions of the initial amplification product as the templates for subsequent amplification. Long-term storage or repeated freeze-thaw cycles will lead to DNA breakage, nicking, or degradation, so the freshly prepared double-stranded DNA should be used as the template. Check whether the extension time is sufficient.Ĭheck whether the synthetic primers have degraded.Ĭheck the quality of the template. If the target fragment is long, try the touchdown PCR program. If the annealing temperature is inappropriate, determine the appropriate annealing temperature by testing a gradient of annealing temperatures. If yeast is used as the template, the pre-denaturation time may be extended to 10 minutes. If the temperature is too low, the template will not be completely denatured. If the temperature is too high, the enzyme will be rapidly inactivated in the first few cycles. Repeat the experiment with the correct system.Ĭheck whether the denaturation temperature is correct and whether the temperature displayed on the PCR system is the same as the actual temperature. The reaction system has been prepared incorrectly. Repeat the experiment with a fresh enzyme or an enzyme from another source. The enzyme used in the reaction has been inactivated. ![]() If cDNA is used, check the purity and integrity of the RNA used for reverse transcription. Crude sample templates may contain inhibitors, so it is recommended to lower the template concentration (use after dilution if a plant leaf is used, make sure the plant is not rich in polysaccharides or polyphenols, take a fresh, young leaf, and trim it to the size of the end of a yellow pipette tip). P021) to effectively lower the melting temperature. An excessively high GC content in the template will make it difficult to separate DNA double strands in this case, add PCR Enhancer (Cat. Long-term storage or repeated freeze-thaw cycles will lead to DNA breakage, nicking, or degradation, so the template should be the freshly prepared double-stranded DNA. Q1: The amplification efficiency is low and there are no amplified bands in the test group.Ĭheck whether the synthetic primers have degraded due to improper storage review primer design and use BLAST to examine primer specificity or redesign primers. The PCR product has an adenine at the 3' end, which can be directly cloned into T vectors and is suitable for ClonExpress and TOPO cloning kits (Vazyme #C112/C113/C115/C601). The kit provides a version containing electrophoresis buffer and green loading dye, which can be directly electrophoresed after the reaction that is convenient to use. The protective agent added to the system allows 2 × Master Mix to maintain stable activity after repeated freezing and thawing. It is suitable for PCR amplification within 5 kb using genome as template and PCR amplification within 10 kb using plasmid and λDNA as template. The kit has excellent amplification performance and high storage stability. The pre-prepared 2 × Master Mix only needs to add primers and templates to perform amplification, which reduces pipetting operations and improves detection throughput and results reproducibility. The maximum amplification speed within 1 kb can reach 1 sec/kb, greatly saving reaction time. The amplification speed of this product can reach 15 sec/kb, which is suitable for rapid PCR. Moreover the initial amplification causes there to be more template of the specific sequence available for amplification also helping to out compete nonspecific products.2 × Rapid Taq Master Mix contains Taq DNA Polymerase, elongation promoting factor, dNTP and an optimized buffer system. By keeping the temperature very close to the melting temperature of the primers initially, the most specific products will be amplified first and thus will not be "swamped out" by the nonspecific products as the temperature of the reaction is lowered. Secondly, the melting temperature of the primers sets an upper limit to the annealing temperature. That is, the lower the temperature, the lower the specificity. First, the specificity of the products is based off the annealing temperature at which the reaction occurs. The procedure is based off of 2 principles. Use this PCR to avoid primers amplifying nonspecific sequences.
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