12/26/2023 0 Comments Alternative to touchdown pcr protocolThe standard reaction protocol and guidelines are largely based on NEB recommendations, and optimization and troubleshooting information are from elsewhere. © 2018 Cold Spring Harbor Laboratory Press. Home Protocols Q5/Phusion PCR Created by Shyam Bhakta, last modified on Q5 DNA polymerase PCR setup, thermocycling, optimization, and troubleshooting are described here. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target-for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, increasing specicity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers. We tried several touchdown PCR programs starting at 60-65 C and ending at 48-50 C, and a step-up PCR program involving five rounds of 10 cycles each stepping up from 48-53 C at 0.5 C. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2☌-5☌ below the calculated T m of the primers. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. In touchdown PCR the temperature selected for the annealing step is initially set 5☌-10☌ higher than the calculated T m of the primers. One of the first alternative methods that we implemented to genotype JAX mice was the High Resolution Melt Curve (HRM) assay. "Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs.
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