![]() Optimizing the annealing temperature of your PCR assay is one of the most critical parameters for reaction specificity. Verify specificity using tools such as the ( Basic Local Alignment Search Tool BLAST).Check the sequence of forward and reverse primers to ensure no 3' complementarity (avoid primer-dimer formation).Avoid repeats of Gs or Cs longer than 3 bases.Avoid secondary structure adjust primer locations so that they are located outside secondary structure in the target sequence, if required.One way to calculate T m values is by using the nearest-neighbor method (use this online T m calculator) Design primers that have a GC content of 50–60%.When designing primers for a PCR assay, follow these guidelines: Choose a region that has a GC content of 50–60%.Avoid regions with long (>4) repeats of single bases.See Real-Time PCR: General Considerations (Bio-Rad bulletin 2593) for more details Use programs such as mfold to predict whether a PCR product will form any secondary structure at the annealing temperature. When possible, avoid regions that have secondary structure.Although short PCR products are typically amplified with higher efficiency than longer ones, a PCR product should be at least 75 bp to easily distinguish it from any primer-dimers that could potentially form See the guidelines under Long PCR Assays when amplifying long sequences. When amplifying any sequence in a given section of DNA for purposes such as genotyping experiments, follow these guidelines to select a product. Commercially available programs such as Beacon Designer software can perform both primer design and target sequence selection. Assess primer properties (melting temperature, secondary structure, complementarity).Ī number of free online resources are available to help you with PCR assay design (see Free Internet Resources for Primer Design).Check the literature and databases for existing primers.When designing primers for a PCR assay, follow these steps: The use of PCR primers specifically designed and validated for PCR assays with your target of interest is highly recommended. Therefore, care must be taken when choosing a target sequence and designing primers. Both the primers and the target sequence can affect the efficiency, specificity, and accuracy of PCR assays. View the PCR Plastics Compatibility Chart to find the right consumables for your instrument.A successful PCR assay requires efficient and specific amplification of the product. Select from our complete line of thermal cyclers using the PCR Thermal Cycler Selection Guide. A gradient-enabled 384-well module for high throughput.A gradient-enabled dual 48/48-well fast module that allows 2 independently controlled protocols to be run side by side in a single bay.The C1000 Touch thermal cycler can be used with six interchangeable reaction modules, including: Thermal gradient - quickly and easily identify optimal annealing temperatures by using the programmable temperature gradient.Convenient file management - keep protocols organized using personalized folders or a USB flash drive.Flexible platform - increase throughput capability by linking up to 3 additional S1000 thermal cyclers or adding PC control for up to 32 cyclers.Easy protocol editing - the large color touch screen and redesigned navigation make editing and running protocols easy.Quick and easy protocol programming - 2 programming options, including the Protocol Autowriter, which automatically generates customized standard, fast, and ultrafast protocols.Each PCR module has a fully adjustable heated lid that supports a wide range of vessels and sealers, including low-profile and standard-height PCR plates. This fully modular platform is able to accommodate different throughput needs with easily interchangeable reaction modules that swap in seconds without tools. The C1000 Touch thermal cycler offers superior performance and a large color touch screen for easy programming.
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