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DNA Polymerase Beta Null Mouse Embryonic Fibroblasts Harbor a Homozygous Null Mutation in DNA Polymerase Iota

DNA Polymerase Beta Null Mouse Embryonic Fibroblasts Harbor a Homozygous Null Mutation in DNA Polymerase Iota

Robert W Sobol. DNA Repair (Amst).

Figures

FIGURE 1
FIGURE 1
PCR genotyping of mouse POLI codon 27 in pol β null and matched wild-type cell pairs. Genomic DNA isolated from each cell line was purified and characterized for the pol ι codon 27 Wt and null allele as described [64]. Briefly, amplification was performed using flanking PCR primers that amplify an 88 bp fragment surrounding codon 27 in a volume of 25 μl, with 250 μM of each dNTP, 10 pmoles of each primer, and 1.25 units of AmpliTaq Gold DNA polymerase. After denaturation at 95°C for 10 min, the reaction mixture is subjected to 35 PCR cycles as follows: denaturation at 95°C for 0.5 min, annealing at 57°C for 0.5 min and primer extension for 1 min at 72°C, followed by a single primer extension step at 72°C for 10 min. The resulting fragment is then purified using a Qiagen PCR spin-column and the DNA is incubated in the presence of TaqI (NEB) at 65°C for 2 hours. The final products were then analyzed by electrophoresis using a 4% NuSieve 3:1 agarose gel in TBE. The amplicon (88 bp) containing a WT allele is digested to two fragments of 39 and 49 bp whereas the null allele (mut) remains as the undigested 88 bp fragment. The cell lines and corresponding genomic DNA used for lanes 1–10 are listed in Table I. M = DNA size markers; WT = DNA derived from C57BL/6 mice harboring a WT POLI codon 27; mut = DNA derived from c129 mice harboring a mutant POLI codon 27.

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