Assembly PCR (CAT#: STEM-MB-0200-WXH)

Assembly PCR essentially involves PCR'ing the two pieces separately with primers that have a 20bp overlap and then doing an extra PCR step using the two products as the template.
Much like how primers are designed such that there is a forward primer and a reverse primer capable of allowing DNA polymerase to fill the entire template sequence, PCA uses the same technology but with multiple oligonucleotides. While in PCR the customary size of oligonucleotides used is 18 base pairs, in PCA lengths of up to 50 are used to ensure uniqueness and correct hybridization.

• Improve the yield of the desired protein.
• Produce large amounts of RNA for structural or biochemical studies.
• Production of synthetic genes.

1.Design the reverse primer for the DNA that will be 5' with significant overlap with the forward primer for the 3' piece.
2.Do PCR as normal for the two (5' and the 3') pieces using the longer primers that correspond to each piece.
3.Check on a gel to make sure you got product from the first PCR reaction.
4.Set up the assembly reaction like a regular PCR.
5.Run the product on a gel.
6.Purify the product