Protein Engineering Protocols - Mycobacteriology research center

Engineered M13 Bacteriophage Coat Proteins 2133.2.2.2. ANNEALING OF THE OLIGONUCLEOTIDE TO THE TEMPLATE1. To the 20 µL of phosphorylation reaction mix, add 20 µg of dU-ssDNA template(from Subheading 3.2.1.), 25 µL of 10X TM buffer, and water to bring to a final volumeof 250 µL. These DNA quantities provide an oligonucleotide to template molarratio of 3:1, assuming that the oligonucleotide to template length ratio is 1:100.2. Incubate at 90°C for 3 min, 50°C for 3 min, and 20°C for 5 min (see Note 5).3.2.2.3. ENZYMATIC SYNTHESIS OF CCC-DSDNA1. To the annealed oligonucleotide and template mixture, add 10 µL of 10 mM ATP,10 µL of 25 mM dNTPs, 15 µL of 100 mM DTT, 30 Weiss U of T4 DNA ligase, and30 U of T7 DNA polymerase.2. Incubate overnight at 20°C.3. Affinity purify, and desalt the DNA using the Qiagen QIAquick DNA PurificationKit. Add 1.0 mL of buffer QG (Qiagen) and mix.4. Apply the sample to two QIAquick spin columns placed in 2-mL microcentrifugetubes. Centrifuge at 14,000g for 1 min in a microcentrifuge. Discard theflow-through.5. Add 750 µL of buffer PE (Qiagen) to each column. Centrifuge at 14,000g for1 min. Discard the flow-through and centrifuge at 13 krpm for 1 min. Place thecolumn in a new 1.5-mL microcentrifuge tube.6. Add 35 µL of ultrapure irrigation USP water to the center of the membrane.Incubate at room temperature for 2 min (see Note 6).7. Centrifuge at 14,000g for 1 min to elute the DNA. Combine the eluants from thetwo columns. The DNA can be used immediately for E. coli electroporation, or itcan be frozen for later use.8. Electrophorese 1.0 µL of the eluted reaction product alongside the dU-ssDNAtemplate. Use a TAE/agarose gel with ethidium bromide for DNA visualization(Fig. 2; see Note 7).A successful reaction results in the complete conversion of dU-ssDNA todsDNA, which has a lower electrophoretic mobility. Usually, at least two productbands are visible and there should be no remaining dU-ssDNA (Fig. 2). Theproduct band with the higher electrophoretic mobility represents the desiredproduct: correctly extended and ligated CCC-dsDNA, which transforms E. coliefficiently and provides a high mutation frequency (~80%). The product bandwith the lower electrophoretic mobility is a strand-displaced product resultingfrom an intrinsic, unwanted activity of T7 DNA polymerase (14). Although thestrand-displaced product provides a low mutation frequency (~20%), it alsotransforms E. coli at least 30-fold less efficiently than CCC-dsDNA. If a significantproportion of the ssDNA template is converted to CCC-dsDNA, a highlydiverse library with a high mutation frequency will result. Sometimes a thirdband is visible, with an electrophoretic mobility between these two productbands (Fig. 2). This intermediate band is correctly extended but unligated