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202 Caldwell et al.5. Targeting screening PCR reaction mixture: 6.3 µL sterile H 2O, 1 µL cresol red, 1 µLbuffer 1, 0.2 µL dNTP, 0.07 µL polymerase mix, 0.2 µL primer forward (25 pmol/µL),0.2 µL primer reverse (25 pmol/µL), and 1 µL DNA from the crude extract.6. Buffer 1 and polymerase mix expand long template PCR system (Roche AppliedScience).3. Methods3.1. Amplification of the ArmsGenomic DNA prepared from wild-type or mutant DT40 cells is used asthe template to generate isogenic fragments for the targeting constructs.Isogenic arms that are identical to the sequence of the target locus were shownto increase the ratio of targeted-to-random integration after transfection ofmurine embryonic stem cells (8). This effect was also seen after transfectionof DT40, although it is most likely less pronounced because of increasedhomologous recombination activity of the cells. Nevertheless, it is advisableto use genomic DNA of DT40 for the amplification of the arm sequencesbecause this may increase the targeting ratio of more difficult genes. As DT40is derived from an out-bred chicken, there remains the chance that one alleleis targeted less efficiently than the other allele because polymorphisms causedifferences between the targeting vector and the allelic locus. It should also benoted herein that chromosome 2 of DT40 exists in triplicate.3.1.1. Genomic DNA PreparationDT40 cells are grown in a humidified CO 2(5%) incubator at 41°C in CM.Day 1:1. Centrifuge 50 mL of healthy viable DT40 cells at 1500 rpm and 4°C for 5 min.The health and viability of the cells is checked with a microscope. The cells shouldhave a rounded shape and build clusters. This is indicative that they are healthyand are in a growing phase.2. Wash the pellet with 1–2 mL 1X phosphate buffer solution (PBS) and centrifugeat 1500 rpm and 4°C for 5 min.3. Resuspend the pellet in 500 µL proteinase K buffer plus 12.5 µL 20% sodiumdodecyl sulfate and transfer to a 1.5-mL tube.4. Incubate tube overnight at 56°C to extract DNA.Day 2:1. Add the same amount of phenol (500 µL) to the DNA extract.2. The mix is rotated gently for 15 min and then centrifuged at 13,000 rpm and 4°Cfor 5min.3. 500 µL of the upper phase are transferred to a new tube containing 500 µLphenol/chloroform (1:1).4. The mix is rotated gently for 15 min and then centrifuged at 13,000 rpm and 4°Cfor 5 min.