Nucleic Acids Research, 2010, Vol. 38, No. 8 2625 Figure 1. Standard assembly versus In-Fusion assembly. (a) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel puriﬁed and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites ﬂanking the parts are maintained. (b) In-Fusion assembly of two BioBricks involves PCR, puriﬁcation, and a subsequent In-Fusion reaction. Parts A and B are PCR-ampliﬁed (in this example the vector is ampliﬁed with Part B) and puriﬁed without gel extraction. Each assembly requires four primers, where two are speciﬁc to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick preﬁx or suﬃx homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The puriﬁed PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites ﬂanking the parts maintain the standard BioBrick format.