Overlap extension PCR (OE-PCR) (CAT#: STEM-MB-0215-WXH)

Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3′ ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3′ region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes.

• Efficient gene cloning and multiple site-directed large fragments insertion, deletion and replacement.
• Site-directed mutagenesis.
• The creation of chimeric molecules .
• Cloning of large gene segments by splicing together smaller pieces.
• Create recombinant plasmids.

1.Primer design
2."Extension PCR": PCR amplify the necessary fragments separately
3.Clean up the product using a DNA column.
4."Overlap PCR": Use cleaned up fragments as template in a PCR reaction
5."Purification PCR": Add end primers to the Overlap PCR reaction
6.Gel extract the correct size fragment.