PCR Primer Design Guidelines
1 Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp This length is long enough for adequate specificity, and short enough for primers to bind easily to the template at the annealing temperature 2 Melting Temperature: Melting Temperature (T m) by definition is the temperature at which one half of the DNA
The polymerase chain reaction - u-szegedhu
has been used up, extra cycles of PCR are required A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction, 12/2004 7 melting temperature of primer-template DNA duplex In most cases the T A is between 50 and 65o C However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 1-2o C Extension
Real-time PCR handbook - Gene-Quantification
The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling In traditional (endpoint)
PRODUCT INFORMATION Polymer Taq DNA Polymerase of reactions
maximal PCR yield The recommended concentration range is 1-4 mM If the Mg2+ concentration is too low, the yield of PCR product could be reduced On the contrary, non-specific PCR products may appear and the PCR fidelity may be reduced if the Mg2+ concentration is too high Taq DNA Polymerase is supplied with two buffers: Taq buffer
Real-Time PCR Applications Guide - Bio-Rad Laboratories
PCR It includes guidelines for designing the best real-time PCR assay for your experiments and explains how real-time PCR data are used in various applications In Sections 5–7, we present sample protocols and data that demonstrate the use of real-time PCR in specific applications, namely, gene expression analysis, allelic
Real Time PCR Ct Values
Real Time PCR Ct Values What does Ct mean? In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level) Ct levels are inversely proportional to the amount of
Introduction to Quantitative PCR - Agilent
Jun 24, 2016 · Real-Time vs Endpoint Quantitative PCR PCR technology is widely used to aid in quantifying DNA because the amplification of the target sequence allows for greater sensitivity of detection than could otherwise be achieved In an optimized reaction, the target quantity will approximately double during each amplification cycle
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