PCR Amplification of Mouse Genomic DNA for Use as Probes

1. Set up a master mix PCR reaction of 175 ^L vol by mixing together 118 ^L deion-ized water, 17.5 ^L of 10X reaction buffer, 14 ^L of 25 m M MgCl2, 3.5 ^L of 10 mM dNTP mix, 7 ^L mouse genomic DNA, and 1 ^L of the 5 U/^L Taq polymerase.

2. Mix well, and divide evenly between two tubes. Add 7 ^L of the 0.4 mg/mL forward primer to one tube and 7 ^L of the 0.4 mg/mL reverse primer to the second tube.

3. Set the thermal cycler for a prewarming time of 5 min at 94°C; a cycling set of 94°C denaturing, 61 to 62°C annealing, and 72°C extension, 35 cycles; and a final cold soak of 4°C. For 24-bp primers designed with IDT's online program, the cal culated Tm is usually roughly 60.0 to 60.5°C, and an annealing temperature of 62°C will usually work well for these primers.

4. Let the two halves of the reaction, in their separate tubes, reach 94°C. Then quickly withdraw the contents (the aqueous portion only when an oil layer is used) of the reverse primer tube and pipet them into the bottom of the forward primer tube, mixing briefly. Let the thermal cycling of the now complete reaction mixture begin.

5. After the cycling is finished, add 5X gel loading buffer to the PCR reaction and run it on a 1.5% agarose gel at 100 V for about 1 h. Stain with ethidium bromide to visualize DNA.

6. Excise the target band of interest and purify from the gel with the Qiaex II extraction kit.

7. Estimate the concentration of the recovered DNA by running a dilution series on an agarose gel along with a lane of DNA size markers of known quantity.

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