PAGE 3 OF 9 repair a range of diﬀerent lesions in vitro. Repair reactions were prepared according to manufacturer’s instructions (NEB) and contained 1.2 mg DNA of the assembly reac- tion in a volume of 150 ml. Quantifying the assembly efficiency by real-time PCR The assembly eﬃciency was quantiﬁed by real-time PCR. A typical ampliﬁcation reaction contained 1 ImmoBuﬀer supplemented with 2 mM MgCl2, 250 mM dNTPs, 1 mM oligonucleotides LMB-2-6 and pIV-B1 (Supplementary Table S1), SYBR Green I diluted 30 000-fold, 1 pg DNA derived from the assembly reaction and 5 U IMMOLASE (Bioline) in a volume of 100 ml. After an initial heat activation and denaturation step for 10 min at 958C, 30 PCR cycles were run as follows: 30 s at 958C, 1 min at 688C and 2 min at 728C. The assem- bly eﬃciency was quantiﬁed using the ‘Comparative Quantiﬁcation’ feature provided with the Rotor-Gene 6000 analysis software (Corbett). Errors are shown as standard deviations of four technical replicates. In Vitro TS–TL from assembled DNA templates DNA templates for in vitro TS–TL were either derived from a purifying PCR (Figure 2, L4 or Supplementary Figure S1, L12) or ampliﬁed from plasmid DNA. When templates were directly ampliﬁed from plasmid DNA, the PCR was run over 25 cycles with 0.5 ng pIVEX-Avi-AGT serving as template in a reaction volume of 100 ml. Extension times were 1 min 30 s and 3 min for Taq and PfuTurbo Cx respectively. A cell-free expression mix was subsequently prepared on ice according to the manufac- turer’s instructions (RTS100, E. coli HY kit, Roche Applied Science) with DNA templates at a ﬁnal con- centration of 5 nM. To label functional Avi-AGT, the in vitro TS–TL reaction was also supplemented with O6-benzylguanine-biotin (BG-biotin) at a ﬁnal concentra- tion of 5 mM. Following expression and conjugation for 4 h at 258C, 4 ml of the TS–TL reaction were cleaned by acetone precipitation, separated by SDS–PAGE on 12% gels and blotted onto nitrocellulose membranes. Membranes were then transferred into TBS-T (20 mM Tris–HCl, 137 mM NaCl, 0.05% Tween 20, pH 7.5), blocked with 2.5% BSA, washed, primed with streptavi- din-horseradish peroxidase (HRP; Sigma, 1:10 000 dilu- tion) and washed. All steps were performed at room temperature under shaking conditions. HRP activity was developed by using ECL detection reagent. Detailed pro- cedures are provided in the Supplementary Data. Assembly of epPCR libraries epPCR libraries of AGT were prepared with the GeneMorph II Random Mutagenesis Kit. Brieﬂy, a typi- cal PCR contained 1 Mutazyme II reaction buﬀer, 200 mM dNTPs each, 0.5 mM oligonucleotides Fw-AGT and Rw-AGT (Supplementary Table S1), 2.5 U Mutazyme II DNA polymerase and 20 ng pIVEX-Avi- AGT in a volume of 50 ml. After an initial denaturation and heat activation step for 2 min at 958C, 30 cycles of PCR were run as follows: 30 s at 958C, 1 min at 628C and 1 min at 728C. This was followed by a ﬁnal extension step Nucleic Acids Research, 2009, Vol. 37, No. 18 e122 for 10 min at 728C. The library was then puriﬁed with QIAquick PCR puriﬁcation kit and subjected to a DpnI digest according to manufacturer’s instructions (NEB). A portion of the library (50 ng or 7 1010 templates in a volume of 100 ml) was then re-ampliﬁed with Taq DNA polymerase under the same conditions described earlier to prepare suitable substrates for the assembly process except that ampliﬁcation featured 25 cycles instead of 30. The re-ampliﬁed library was then precipitated with PEG– MgCl2, blunted with T4 DNA polymerase, assembled and puriﬁed by PCR. For sequencing, individual DNA templates were cloned into a pCR 2.1 vector using the TOPO TA cloning kit according to manufacturer’s instructions (Invitrogen). Model affinity selections Model aﬃnity selections were performed as previously described with slight modiﬁcations to accommodate for the selection of biotinylated protein–DNA conju- gates (27). Detailed procedures are provided in the Supplementary Data. RESULTS Assembly strategy A template regeneration strategy is presented that over- comes many of the practical shortcomings of the existing assembly strategies (Figure 1). The GOI is assembled into a functional template for in vitro TS–TL with USER Enzyme (22–24) and T4 DNA ligase. USER Enzyme is a commercially available enzyme mixture composed of uracil DNA glycolyase (UDG) and endonuclease VIII. In combination, the two enzymes excise uracil nucleotides from DNA thereby leaving a single base pair gap. If a uracil residue is incorporated close to the 50-end of a linear DNA fragment by PCR and the resulting 50-oligonucleotide can eﬃciently dissociate, single- stranded 30-extensions are generated that subsequently direct the assembly of DNA fragments through comple- mentary, overlapping ends. In the presence of T4 DNA ligase the assembled fragments are then covalently sealed which makes the desired product template amenable to ampliﬁcation by PCR. Assembly and PCR purification Coupled uracil excision–ligation is eﬃcient as the majority of substrate DNA fragments assembled into the desired product in 10 min (Figure 2, L3, Supplementary Figure S1). Suitable substrate DNA fragments with a uracil residue near the 50-end were either prepared with Taq or PfuTurbo Cx (28). These constitute the only two commercially available thermostable DNA polymerases that can eﬃciently replicate uracil-containing DNA tem- plates (24,28). For eﬃcient assembly, it was crucial that templates were treated with T4 DNA polymerase in order to remove the 30-adenine overhangs generated by the extendase activity of Taq. While PfuTurbo Cx generates blunt ends, we observe variable results in the assembly eﬃciency so that it may become necessary to blunt the