Protein Engineering Protocols - Mycobacteriology research center

244 Ghadessy and Holliger2. Prepare oil-phase CSR by mixing light mineral oil with 4.5% (v/v) Span 80, 0.4%(v/v) Tween-80, and 0.05% (v/v) Triton X-100, under constant stirring at roomtemperature. Because of the high viscosity of the surfactants, it is advisable to preparea large volume of oil-phase CSR (>50 mL) and use cut-off pipet tips to dispensethe surfactants. The oil-phase CSR, once prepared, can be stored in the darkat room temperature for 1 mo.3. Add 200 µL of aqueous-phase CSR mix drop-wise (~5–10 µL per drop, one dropevery 5 s) to 400 µL of oil-phase CSR in a 2-mL round-bottom Biofreeze vialunder constant stirring (1000 rpm) with a magnetic stir bar.4. After addition of the last drop, continue stirring for 5 min. The emulsion shouldbe creamy white and viscous (see Note 2).3.4. CSR1. Transfer the emulsion mix to 0.5-mL thin-walled PCR tubes (100 µL/tube) andadd two drops of mineral oil to prevent evaporation (see Note 3).2. Carry out PCR thermocycling, typically using 20 cycles with the profile 94°C(1 min), 60°C (1 min), 72°C (5 min), preceded by an initial 5-min incubationat 94°C to lyse bacterial cells and destroy background enzymatic activities.After thermocycling, the emulsion phase should remain creamy white andshould be overlaid with a clear oil phase. The volume may have reduced but thisdoes not indicate coalescence of the emulsion compartments. Coalescence andbreaking up of the emulsion manifests itself by the appearance of a clear layerof aqueous phase just beneath the white emulsion phase. This may indicate thepresence of a variety of factors that interfered with the creation of a stable emulsion,including, for example, insufficient mixing.3.5. Work-Up1. Recover the aqueous-phase CSR by extraction of the emulsion with a double volume(200 µL) of diethyl ether. Mix by vortexing at maximum speed for 20 s, andspin at 20,000 rcf (13 krpm) for 2 min in a benchtop centrifuge.2. Two liquid layers should be apparent. Carefully remove the lower, often cloudy,aqueous phase from underneath the clear organic phase and transfer into a fresh1.5-mL Eppendorf tube.There are two different methods for the workup of CSR reactions that wehave found to work.3.5.1. PEG Extraction Method1. Extract the aqueous phase once with phenol–chloroform (1/1 v/v), and then againwith chloroform–isoamyl alcohol (24/1 v/v) alone.2. To the aqueous phase, add 0.5 volumes of PEG 800/MgCl 2solution (see Subheading2., item 13) and mix well by pipetting up and down carefully several times.