![]() ![]() For example, in PCR/qPCR, the target concentration is usually designed to be much lower than that of the probe. ![]() The strand that is in excess determines T m. Oligo concentration alone can cause T m to vary by ☑0☌. ![]() While folding of a single oligonucleotide is concentration independent, the T m is strongly influenced by oligo concentration when 2 or more nucleic acid strands interact. T m varies with oligonucleotide concentration. Additional factors must be considered, such as oligo concentration and the environment in which hybridization will take place.” Important factors that affect T mare discussed in the following sections. “ T m is not a constant value, but is dependent on the conditions of the experiment. “ But this is not how T m works,” says Dr Owczarzy. Melting temperature (Tm) explainedĭr Richard Owczarzy (Ohv-char’-zee), a senior researcher at IDT and an extensively published expert on nucleic acid thermodynamics, confirms that many researchers are still under the illusion that they can use a simple formula to calculate T m-multiplying the number of GC and AT bases by a given constant. It is very important to understand this process so that you can better design and optimize the oligos for your experiments. Therefore, an understanding of melting temperature (T m) provides information on when and how the DNA or RNA strands are going to hybridize and defines the rules for hybridization. The criteria for hybridization are based on nucleic acid strand melting. Examples of techniques that include nucleic acid hybridization are northern and Southern analysis, PCR/qPCR, cloning, in situ hybridization, array analysis, gene knockdown, and next generation sequencing (NGS). Hybridization is a common step of many, if not most, molecular biology protocols.
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