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208 Caldwell et al.PCR amplify the extract using the targeting screening reaction mixture:Use various primer pairs to identify the targeted clones (refer to Fig. 4):1. Primer forward upstream of 5′ arm and primer reverse inside the drug resistancemarker cassette to amplify the marker and to exclude single crossing over.2. Primer forward and primer reverse inside target locus to amplify the deleted region.3. Check PCR screen through agarose gel electrophoresis.4. Select a number of targeted clones and grow in 24-well flat bottom microtiterplates by adding 100 µL of the correct cell suspension from the 96-well flat bottommicrotiter plate and add 2 mL CM (see Notes 8 and 9).5. Select an appropriate clone and repeat the transfection procedure using a secondtargeting construct with a different marker cassette.At this point, it is highly recommended to perform the phenotype experimentsand consider the results in selecting specific heterologous knockoutclones for further use.4. Notes93°C 2 min93°C 30s65°C 30s68°C 5min*68°C 7min4° C ∞⎫⎪⎬⎪⎭35 cycles*time increases: 20 s each cycle1. Use only trusted suppliers of cell culture reagents and always test new lot numbersas well as each batch of freshly made CM.2. The addition of DMSO is known to help with difficult spots and it is used in thesequencing protocol wherein its’ inclusion increases both the quality and the lengthof the sequencing product. Glycerol has sometimes been used for PCR, but in theoriginal tests that were done on it previously, it was found that additives such asIpegal and DMSO performed better. This effect was seen with multiple polymerasesincluding Taq, TaqGold, Vent, MMLV-RT, and Superscript RT.3. Marker recycling:a. The marker cassette can be removed from the knockout clones to exclude sideeffects of the drug resistance marker and to reuse the marker.b. Cells with viability over 80% are diluted to a concentration of 0.6 × 10 6 cellsin 2 mL.c. Add 100 µL 4-tetrahydroxytamoxifen (40 µM) to the 2 mL of cells (final concentration2 µM) and incubate for 5–12 h at 41°C. The concentration and timingof this step will have to be empirically determined based on the targeted locus.