The overall success of OE-PCR based DNA assemblies relies on several factors, being the most relevant ones the instrinsic features of the DNA sequence to assemble, the sequence and length of the overlapping overhangs, the design of outer primers for the final amplification and the conditions of the PCR reaction. Normally, from 2 to 6 fragments can be spliced simultaneously into a single OE-PCR reaction. Overhangs should be at least 40 nucleotides long to ensure adequate interaction between fragments. Final amplification primers are commonly designed following general guidelines for PCR, however they are used in 2 to 5 times lower concentration than in standard PCR reactions, as it this has been shown to reduce undesired amplifications. Additionally the utilization of proofreading DNA polymerases is highly recommended.