![]() ![]() This can save time and money while waiting for sequencing info on the putative mutated clones. If you can introduce a novel restriction site through a silent mutation, you can screen the mutated plasmids by digest. The formula provided in the kit information will often yield a different melting temperature than that calculated by standard methods be sure to use the formula suggested by the kit to get the right primer length and composition. ![]() Use the company’s Tm formula to calculate the melting temperature.Here are a few additional things to keep in mind: Mutagenesis kits contain extensive information on how to design optimal primers for mutagenesis, which can vary significantly from cloning primers in length, composition, and melting temperature. Primer-based mutagenesis is a quick and easy way to mutate a gene of interest. Since the extra bases do not anneal to the template, they do not need to be included in the melting temperature calculation. This will increase the efficiency of the restriction reaction (by giving the enzyme something to “hold on to”). Add a few extra nucleotides upstream of restriction sites.Any annealing between your primer pair or of one primer to itself will drastically affect the efficiency of your PCR reaction. If both primers have a melting temperature within 1-2 degrees of each other, it will be much simpler to optimize your PCR conditions. Match the melting temperatures of paired primers.The G-C bond is stronger than the A-T bond, so a 3’ end enriched in G and C residues will bind with higher affinity. Use a “GC clamp” at the 3’ end of each primer.A few tweaks, however, can increase the chance of cloning your gene on the first try: The desired amplicon and the vector restriction sites will largely dictate the composition of the primers. One of the most common primer-based applications is cloning. Here are a few tips on optimizing primer design for several different applications: PCR amplification/cloning Primer design can sometimes feel like more of an art than a science, and designing the best primer can significantly affect the success or failure of your experiments. ![]()
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