One-step generation of error-prone PCR libraries using Gateway® technology

Table 5 Comparison of expression library complexities provided by different combinations of epPCR products and of non-linearized acceptor constructs

epPCR product	Non-linearized acceptor construct	Number of clones
N_TAIL¹ 10 fmoles	pNGG⁴	2.8 × 10⁷
N_TAIL¹ 100 fmoles	pNGG⁴	3.25 × 10⁷
LadS² 10 fmoles	pTH31⁵	6.65 × 10⁷
LadS² 100 fmoles	pTH31⁵	3.85 × 10⁷
PA3059³ 42 fmoles	pTH31⁵	1 × 10⁷

The number of E. coli colonies provided by various combinations of epPCR product and of 25 fmoles of acceptor constructs in 5 μl of LR reaction is reported. T7pRos E. coli cells were electroporated and spread on ACplates
¹N_TAIL PCR product is 661 bp. ²LadS PCR product is 696 bp. ³PA3059 PCR product is 1,780 bp. These three PCR product lengths include the att L1 and att L2 recombination sites. ⁴pNGG is 8,012 bp before LR recombination. ⁵pTH31 is 8,306 bp before LR recombination.

ISSN: 1475-2859