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Primers for crossover PCR should include an overhang with a restriction enzyme cutting site. In general, about 1.5 kb PCR-amplified fragments work well for crossover PCR (XO-PCR).

Examples of XO-PCR primers that include a HindIII site:
Primer B: CGT ACT CTG CGA AGC TTC C
Primer C: GGA AGC TTC GCA GAG TAC G
Make sure that the enzyme site on your primer is not also in your PCR-amplified fragment.
Amplify the AB and CD regions separately (See figure).




