Help:Primers/Construction

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Once you've designed a primer, you can then order it via from a oligo synthesis facility at your institution or a commercial company. Chemical synthesis occurs via sequential addition of bases from 3' to 5', and addition of each base is approximately 99% efficient. Thus, when you receive a synthesized oligo, only 0.99n-1 of the molecules will be correct, where n is the primer length. For example, for a primer of length 70 nucleotides, only 50% of the molecules will be correct. Errors in primer synthesis include truncations, internal deletions, and mutated bases. Truncated primers or primers with internal deletions can be eliminated via purification by HPLC or PAGE. Such purification steps are available from most oligo synthesis companies, but do substantially increase primer cost. For primers over over ~60 nucleotides in length or for sensitive applications, such as terminator construction, you might consider paying for an extra purification step when ordering a primer.

Primers sequences should always be specified from 5' to 3' when placing orders. Generally, this means that the "forward" primer sequence can be taken directly from the template sequence, whereas the "reverse" primer should be the reverse complement of the template sequence. All primer sequences in the Registry are specified from 5' to 3'.

References