e122 Nucleic Acids Research, 2009, Vol. 37, No. 18 time and can also have unexpected detrimental eﬀects on downstream applications caused by salt and residual agar- ose contaminants (7,17,18). Along with time, conventional methods are inherently complex and, therefore, susceptible to accidental errors. For instance, suboptimal template regeneration in one cycle of a multistep selection process may quickly diminish the size of a library by several orders of magnitude, thereby potentially cancelling out the advan- tage of screening large libraries. Considering the intrinsic ampliﬁcation power of PCR, suboptimal assembly may not necessarily be obvious to the experimenter at the time, especially since it is generally not possible to monitor the integrity of an assembly process using simple means such as agarose gel electrophoresis, and instead requires more sophisticated methods such as competitive (13) or real-time PCR. Here, a simple and eﬃcient protocol is presented that allows the assembly of a gene with a peptide tag and its ﬂanking untranslated regions (UTRs) in 90 min (Figure 1). Assembly is based on a coupled uracil excision–ligation strategy that is reminiscent of UDG (19–21) and USER enzyme cloning (22–24), but also includes T4 DNA ligase to generate DNA templates devoid of nicks. Pure templates that do not need to be puriﬁed further by agarose gel electrophoresis and that are functional for in vitro TS–TL are obtained following a short PCR over 10 ampliﬁcation cycles. The procedure is exempliﬁed for a mutant of O6-alkylguanine alkyltransfer- ase (AGT) (25), which is assembled with an N-terminal peptide tag and its 50- and 30-UTRs that carry all the nec- essary components for in vitro TS–TL including a T7 pro- moter, a ribosome-binding site and a T7 terminator. The utility of the method is further corroborated by assem- bling error-prone PCR (epPCR) libraries and regenerating templates following model aﬃnity selections. The method is suitable for screening and selection systems of very high throughput; up to 1011 molecules can be eﬃciently assembled and puriﬁed in reaction volumes of 100 ml. MATERIALS AND METHODS General General procedures, reagents, sequences of the oligonu- cleotides and the fully assembled Avi-AGT gene integrated into a pIVEX backbone are listed in the Supplementary Data. Preparation of assembly substrates The assembly substrates (50-UTR-Avi, 30-UTR and AGT) were prepared by PCR using BioTaq DNA polymerase. A typical PCR contained 1 NH4-based reaction buﬀer [60 mM Tris–HCl, 6 mM (NH4)2SO4, 10 mM KCl, pH 8.3] supplemented with 2 mM MgCl2, 250 mM dNTPs, 2 mM oligonucleotides each (Supplementary Tables S1 and S2), 0.5 ng pIVEX-Avi-AGT and 5 U BioTaq in a volume of 100 ml. After an initial denaturation 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 for 1 min at 728C. To prevent repeated car- ryover contamination with wild-type DNA from the PAGE 2 OF 9 source plasmid, the PCRs of the ﬂanking regions (50-UTR-Avi and 30-UTR) were digested with 40 U DpnI per 50 ml PCR for 20 min at 378C. Alternatively, AGT was prepared with PfuTurbo Cx. In this case, a typical PCR contained 1 PfuTurbo Cx reaction buﬀer, 250 mM dNTPs, 1 mM oligonucleotides each (Supplementary Tables S1 and S2), 0.5 ng pIVEX-Avi-AGT and 5 U Pfu- Turbo Cx in a volume of 100 ml. After an initial denatura- tion 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 1.5 min at 728C. This was followed by a ﬁnal exten- sion step for 10 min at 728C. PEG–MgCl2 precipitation of assembly substrates To remove any primer dimers and other small non-speciﬁc by-products, all assembly substrates were puriﬁed by pre- cipitation with PEG–MgCl2 (26). Brieﬂy, a solution of DNA at a ﬁnal concentration of 25–50 ng/ml in 10 mM MgCl2, 2–5 mM Tris–HC1, pH 8.5 and varying concen- trations of PEG-8000 was spun at 16 000g for 10 min. The supernatant was then carefully removed by pipetting and the pellet resuspended in 10 mM Tris–HCl, pH 8.5. Yields were typically 60–70%. For optimal results, the concen- tration of PEG-8000 was titrated for each DNA fragment: 10% for 30-UTR of 239 bp, 9.1% for 50-UTR-Avi of 410 bp and 8.3% for AGT of 638 bp. See Supplementary Data for further details on titrating the optimal concentra- tion of PEG-8000. Where applicable, assembly substrates were blunted with T4 DNA polymerase according to the manufacturer’s instructions (NEB). Coupled uracil excision–ligation A typical assembly reaction contained 1 T4 DNA ligase buﬀer, 3 pmol AGT, 4.5 pmol 50-UTR-Avi, 4.5 pmol 30-UTR, 3 U USER enzyme (equivalent of 1 U/1 mg DNA) and 600 U T4 DNA ligase (equivalent of 200 U/ 1 mg DNA) in a volume of 100 ml. The reaction mixture was incubated for 10 min at 378C. A formula is provided in the Supplementary Data to perform mass-to-molar conversions. Purifying PCR Template puriﬁcation was performed with BioTaq DNA polymerase. A typical ampliﬁcation reaction contained 1 NH4-based reaction buﬀer [60 mM Tris–HCl, 6 mM (NH4)2SO4, 10 mM KCl, pH 8.3] supplemented with 2 mM MgCl2, 250 mM dNTPs, 1 mM oligonucleotides LMB-2-6 and pIV-B1 (Supplementary Table S1), 50–500 ng DNA of the assembly reaction as indicated and 5 U BioTaq in a volume of 100 ml. After an initial denaturation step for 2 min at 958C, 10 PCR cycles were run as follows: 30 s at 958C, 1 min at 688C and 2 min at 728C. 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). Template repair DNA templates were repaired with the PreCR Repair Mix, which contains a cocktail of enzymes that can