PCR

PCR product size

Locate the primer binding sites on a template, size the amplicon and check specificity

Input sequence

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Forward primer Reverse primer

Matching Anchor length Sequence is circular

—Product length

—Product GC

—Span on the template

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Colony PCR: telling positives from negatives

Enter the insert length to see whether positive and negative colonies give products you can tell apart on a gel. If the empty vector carries a stretch between the primers that cloning removes, put its length in the second field.

Insert length bp Vector stretch removed bp

—Product from a positive colony

—Product from a negative colony

—Difference

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The search demands an exact match and tolerates no mismatches. Primers carrying a tail — a restriction site or a tag — will not be found in whole-primer mode; use 3′ end matching, which is the part that actually anneals to the template.