I need to clone the lac promoter from the E. coli K-12 strain, which is 31 bp long.
Amy Klocko
You could clone it. However, you may as well just order the 31 bp fragment as a pair of primers and anneal them.
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Bertrand Cornu
Design the oligonucleotide: The first step in introducing an oligonucleotide by PCR mutagenesis is to design the oligonucleotide. The oligonucleotide should be complementary to the region of the DNA template that you want to mutate, and should contain the desired change or modification. It is generally recommended to use a software tool such as Primer3 to design the oligonucleotide and ensure that it is properly synthesized. Prepare the PCR reaction: Once the oligonucleotide has been designed, you will need to prepare the PCR reaction. This typically involves adding the DNA template, the oligonucleotide primers, nucleotides, and a suitable polymerase to a PCR tube. It is important to ensure that the reaction is set up properly and that all of the necessary components are present. Perform the PCR reaction: The PCR reaction can then be performed using standard PCR conditions. This typically involves multiple cycles of heating and cooling to denature, anneal, and extend the DNA template. The oligonucleotide primers will bind to the template DNA and serve as primers for the polymerase to synthesize the new DNA strand. Purify the PCR product: Once the PCR reaction is complete, the PCR product can be purified using a suitable purification method, such as enzymatic ExoSAP. The purified PCR product can then be used as a template for further experiments, such as DNA sequencing to confirm the desired mutation.
PCR mutagenesis is a widely used method for introducing specific changes or modifications into a DNA sequence. To introduce your 31 bp fragment by PCR mutagenesis, you can follow these general steps:
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