Designing Oligonucleotide Primers for PCR

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Designing Oligonucleotide Primers for PCR

(a) Obtain the sequence of the template (either by performing molecular cloning of the gene of interest,  finding the sequence in a database, or sequencing the template).
(b) Design one pair of oligonucleotide primer for each gene or sequence to be amplified by PCR.
(c) Each oligonucleotide primer should be approximately 15-50 bases in length.
(d) The first primer of each pair should cover the 5' end of the gene
(e) The second primer should be the reverse complement of the 3' end of the gene, but the sequence should be written in the 5' to 3' direction when ordering new primers.

To design suitable oligonucletide primers for PCR, it is also important to check if the oligonucleotide primers have the following characteristics:

  1. Are annealing temperatures of each pair of primers for a corresponding PCR product similar?
  1. Does the primer have
    1. An annealing segment?
    2. A tag segment?
    3. A restriction enzyme site?
    4. Extra bases to help cut the restriction site?
  1. Is the primer in the correct orientation (if necessary, is it in reverse complement?  If necessary, are the extra bases [d] in reverse complement?)
  2. Is the annealing temperature at least 50 degrees Celsius?

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