Figure 3

Effects of homology and insert length upon the efficiency of ABI-REC. (A) Homology was varied by increasing the length of P1R primer in the asymmetric bridge PCR reaction. The reactions with 20 bp and 25 bp P1R primers gave rise to maximum output of fused fragments. (B) Transformation of equal volumes of these PCR products into E.coli cells produced double resistant colonies with various numbers. Colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against homology. Error bars indicate mean ± SD from three independent assays. (C) Inserts ranging from 1.6 kb to 4 kb were fused into pUC19 in asymmetric PCR reactions. Red arrows denote the fused fragments. (D) Transformation of equal volumes of these PCR products into E.coli cells produced various numbers of double resistant colonies. Quantitation of single colonies revealed that colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against insert size. Error bars indicate mean ± SD from three independent assays. (E) ApaI and SalI restriction digestion of the plasmids extracted from the single colonies in (D). All plasmids released the inserts as designed. Please note that 2.5 kb insert is nearly identical to backbone pUC19, as one band was observed. In addition, the 4 kb insert has three ApaI sites, one of which is only 133 bp in size and therefore undetectable in the gel. A 367 bp band is denoted by a red arrow.