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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 "Typical" Cycling Conditions
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.
When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10). Extension: The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended. Cycle number: Generally, 25–35 cycles yields sufficient product.
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli , etc.) or complex templates < 1 kb.
PCR (Polymerase Chain Reaction) PCR Troubleshooting. In conventional PCR, problems with reaction components and amplification protocols are diagnosed by running a gel.
Select an extension time suitable for the amplicon length. Reduce the extension temperature (e.g., to 68°C) to keep the enzyme active during amplification of long targets (e.g., >10 kb). Use DNA polymerases with high processivity for robust amplification even with short extension times.
—recommended extension time. <45 cycles. —to avoid nonspecific bands and accumulation of byproducts. Pro tip. Direct PCR vs. multiplex PCR. Direct PCR amplifies target DNA directly from. samples without nucleic acid isolation, vastly simplifying workflow and preventing DNA loss during purification.
An effective PCR experiment requires adequate knowledge of each reaction component and step-by-step procedure to attain the optimized results.
Changing Reaction Time or Amounts. In terms of reaction time, research has suggested that increasing the extension time in a PCR experiment can help amplify longer and more specific DNA sequences while maintaining efficiency.
Which extension temperature should I use, 68°C or 72°C? What is the optimal amount of DNA template that should be used for PCR? What are the critical factors for amplification of long genomic targets? How do I determine if a template is GC rich? What are the critical factors for amplification of GC-rich templates?