Protein Engineering Protocols - Mycobacteriology research center

Monobodies 101and 100 µL of fresh E. coli. Vortex and immediately pour into an LB plate.Incubate the plates at 37°C overnight and titer.3.1.3. Preparation of Mutagenic Oligonucleotides1. Design an oligonucleotide that contains the 5′ and 3′ complimentary regions ofapprox 20 bases and approx 15 bases, respectively, depending on the GC content.We use the NNK or NNS codons for “hard” randomization, where N denotes anequimolar mixture of all nucleotides; K denotes an equimolar mixture of G and T,and S denotes an equimolar mixture of G and C.2. Run approx 10 µg of a crude oligonucleotide dissolved in water on acrylamide gel(use ~5 cm for each 0.1-cm well), cut out a band that corresponds with the correctsize, and electroelute the oligonucleotide (see Note 3).3. Mix 2 µg purified oligonucleotide, 3 µL T4 polynucleotide kinase buffer, 5 U T4polynucleotide kinase (New England Biolab), 1.5 µL 10 mM adenosine triphosphate,and water to bring up to 30 µL. Incubate at 37°C for 2 h and then at 65°Cfor 15 min. Store the solution at –20°C.3.1.4. Synthesis of Double-Stranded DNA1. Mix approx 1 µg of uracil-single-stranded DNA, 2 µL of phosphorylated oligonucleotide(Subheading 3.1.3.), 1 µL of 10X annealing buffer (Bio-Rad Mutagenekit), and bring up to 10 µL with H 2O (see Note 4). Prepare a control reaction withoutan oligonucleotide.2. Using a polymerase chain reaction (PCR) machine, incubate at 70°C for 5 min andthen decrease the temperature to 30°C, at a rate of –1°C/min. Place the tube on ice.3. To the annealing mixture, add 1 µL of 10X synthesis buffer (Bio-Rad Mutagenekit), 1 µL of T4 DNA ligase, and 1 µL of T7 DNA polymerase (New EnglandBiolabs). Incubate for 5 min on ice, 5 min at room temperature, 30 min at 37°C,and 15 min at 75°C, and then cool to room temperature. Run 1 µL of the sampleon agarose gel. The sample with the oligonucleotide should yield products ofhigher molecular weights than the control without an oligonucleotide.4. Dialyze the synthesis mixture by placing a drop of the mixture on a 0.025-µmmembrane disk floating on water for 1 h, then recover the dialyzed mixture in afresh microcentrifuge tube. Keep it on ice until use.3.1.5. Preparation of Electrocompetent Cells1. Grow E. coli SS-320 in 350 mL of superbroth containing Tc until the OD 600nmreaches 0.8.2. Chill the culture in the flask quickly in ice-water bath, then keep it on ice for 15 min.Transfer the culture into a centrifuge bottle and centrifuge at 0 to 2°C at 3900g(4.7 krpm in a JA-10 [Beckman] or equivalent rotor) for 5 min (see Note 5).3. Discard the supernatant and suspend cells in 10 mL HEPES buffer by knockingthe centrifuge tube on ice. Add 390 mL HEPES buffer, and swirl to suspend cells.Centrifuge at 0 to 2°C at 3900g for 5 min.