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206 Caldwell et al.3.1.6. Plasmid Preparation and RE AnalysisOnce the targeting constructs have been successfully built and confirmed,a large-scale plasmid preparation will need to be performed to obtain enoughplasmid for transfection into DT40 (40 µg are needed per transfection).Using one’s favorite large-scale plasmid preparation method, determine theconcentration and dilute to 1µg/µL. This is the final step before transfectionand it is extremely important that one be sure of the product in hand. Sowhen in doubt, repeat RE analysis or do partial sequencing to be assured ofwhat one is working with.3.1.7. Linearization1. The plasmid is linearized using a single cutter, in this case NotI.2. Digest overnight at 37°C in linearization mixture.The following day:1. Add to the overnight digest 1 vol phenol–chloroform (400 µL). Mix well andcentrifuge at 13,000 rpm for 5 min at 4°C.2. Transfer the upper phase to a new tube containing 1 vol chloroform (400 µL). Mixwell and centrifuge at 13,000 rpm for 5 min at 4°C.3. Transfer the upper phase to a new tube containing 1 vol isopropanol (400 µL) andadd 0.1 vol 3 M sodium acetate (40 µL). Mix well and centrifuge at 13,000 rpmfor 30 min at 4°C.4. Discard the supernatant and wash the pellet with 80% ethanol. Do not mix.Centrifuge at 13,000 rpm for 5 min at 4°C.5. Discard the supernatant under a laminar flow hood and dry the pellet 10–20 min.6. Add 300 µL sterile H 2O for an end concentration of 1 µg/µL and store at 4°C fora minimum of 1 h to resolve the pellet, which can be used immediately or storedat –20°C.3.2. Knockout3.2.1. Transfection of DT40 CellsThe construct is now ready to be transfected into DT40 to begin the knockoutprocess.1. Perform a cell viability count on DT40 cells grown in CM. It should be noted thatsome DT40 mutants have a considerably lower viability than wild-type and oneshould use the healthiest possible cells regardless.2. Take 5 × 10 6 viable cells and centrifuge for 5 min at 1500 rpm and 4°C.3. While centrifuging the DT40 cells prepare a 1-mL electroporation cuvet; add40 µL of the linearized construct (concentration 1 µg/mL) and place cuvet on ice.4. Resolve the DT40 cell pellet in 800 µL sterile 1X PBS or CM, add 800 µL to each cuvet.