How to: Design PCR primers and check them for specificity

Starting with ...

ONE OR MORE PRIMER SEQUENCES

  1. Go to the Primer BLAST submission form.
  2. Enter one or both primer sequences in the Primer Parameters section of the form. If only one primer is available, a template sequence is also required. See "A Target Template Sequence..." below.
  3. In the Primer Pair Specificity Checking Parameters section, select the appropriate source Organism and the smallest Database that is likely to contain the target sequence. These settings give the most precise results. For broadest coverage, choose the nr database and do not specify an organism.
  4. Click the "Get Primers" button to submit the search and retrieve template and specificity information.

A TARGET TEMPLATE SEQUENCE OR ACCESSION NUMBER

  1. Go to the Primer BLAST submission form.
  2. Enter the target sequence in FASTA format or an accession number of an NCBI nucleotide sequence in the PCR Template section of the form. If the NCBI mRNA reference sequence accession number is used, the tool will automatically design primers that are specific to that splice variant.
  3. If one or both primer sequences are to be used in the search, enter these in the Primer Parameters section of the form. Primer BLAST performs only a specificity check when a target template and both primers are provided.
  4. In the Primer Pair Specificity Checking Parameters section, select the appropriate source Organism and the smallest Database that is likely to contain the target sequence. These settings give the most precise results. For broadest coverage, choose the nr database and do not specify an organism.
  5. Click the "Get Primers" button to submit the search and retrieve specific primer pairs.
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