Protein Engineering Protocols - Mycobacteriology research center

212 Sidhu et al.Fig. 2. In vitro synthesis of CCC-dsDNA heteroduplex. The reaction products wereelectrophoresed on a 1.0% TAE/agarose gel containing ethidium bromide for DNAvisualization. Lane 1: 1-kb DNA marker (Gibco BRL); lane 2: the dU-ssDNA template;lane 3: reaction product from Subheading 3.2.2. The lower band is correctly extendedand ligated CCC-dsDNA, the middle band is nicked dsDNA, and the upper band isstrand-displaced dsDNA.3.2.2. In Vitro Synthesis of Heteroduplex CCC-dsDNAA three-step procedure is used to incorporate the mutagenic oligonucleotideinto heteroduplex CCC-dsDNA, using dU-ssDNA as a template. Theprotocol described here is an optimized, large-scale version of a previouslydescribed method (13). The oligonucleotide is first 5′-phosphorylated(Subheading 3.2.2.1.) and then annealed to a dU-ssDNA template(Subheading 3.2.2.2.). The oligonucleotide is enzymatically extended andligated to form heteroduplex CCC-dsDNA (lane 3 in Fig. 2), which is thenpurified and desalted (Subheading 3.2.2.3.). This protocol produces approx20 µg of highly pure, low-conductance CCC-dsDNA. This is sufficient forthe construction of a library containing more than 10 10 unique members (seeNote 3).3.2.2.1. OLIGONUCLEOTIDE PHOSPHORYLATION WITH T4 POLYNUCLEOTIDE KINASE1. In a 1.5-mL microcentrifuge tube, combine 0.6 µg of the mutagenic oligonucleotide,2.0 µL of 10X TM buffer, 2.0 µL of 10 mM ATP, and 1.0 µL of 100 mMDTT. Add water to bring to a total volume of 20 µL.2. Add 20 U of T4 polynucleotide kinase. Incubate for 1.0 h at 37°C (see Note 4).