Edited by Chang Zhu
1. Primers specific to the probe is used to amplify DNA from the
2. Two probe-specific primers (primer 2 and 3) are combined with
phage specific primers (primer 1 and 2) in two separate PCR reactions
to amplify the left (primer 1/2) and right (primer 3/4) fragments of the insert.
3. 1 ml of phage (108 pfu)
may be PCR amplified in a 25 or 50 ml PCR reaction.
4. After the two fragments have been successfully isolated, the full
length insert can be generated by annealing the two PCR products and
amplify using the phage specific primers (primer 1/4).
5. Dilute (e.g., 1:5-1:100) or remove probe-specific primers by purification, and
mix equal molar amount of the two PCR fragments. PCR using the
phage specific primers.
6. Multiple pairs of the probe-specific primers may be used to confirm
See also PCR construction of insertion/deletion/mutation/fusion