Genotyping Animals

The following is a one-tube method for preparing genomic DNA from tail biopsies. To obtain uniform signals, it is important to apply enough DNA to saturate the membrane. This was determined empirically using a Bio-Rad 96-well dot-blot apparatus and Amersham Hybond N+ membrane. Figure 2 shows the results of a typical dot blot from intercrosses of a line of mice carrying a single-copy insertion. Homozygous mice, which in this case carry two copies of the vector, are easily distinguishable from their heterozygous litter mates.

1. Tail biopsies (1.5 cm in length) are taken at weaning age and digested overnight at 55-65°C in 0.4 mL of tail buffer.

2. While tubes are still warm, add 0.1 mL 5 M NaCl and vortex at high speed for 5-10 s. Add 0.5 mL of chloroform, and vortex again for 5-10 s. Spin in microfuge for 5 min.

3. Transfer 50 | L of the top aqueous phase to a 96-well plate. Denature DNA by adding 150 |L of 0.53 M NaOH and incubating at 37°C for 30 min.

Fig. 2. Genotyping mice by dot-blot analysis. Tail DNA from intercross litters carrying a single-copy gene trap insertion were hybridized to a lacZ probe. Dots showing weak signals represent heterozygous animals, whereas those showing signals of twice the intensity represent homozygotes.

4. Apply samples to dot-blot apparatus, and leave for 30 min before applying a vacuum.

5. Prepare the dot-blot apparatus by cutting a piece of Hybond N+ membrane and a piece of Whatman paper to fit the apparatus. Prewet the membrane in H2O, and then soak in 0.4 M NaOH for 10 min. Prewet the Whatman in 0.4 M NaOH, and place it underneath the membrane on the apparatus.

6. Draw samples through with gentle vacuum. Disassemble apparatus and wash membrane with 30 mM NaP/0.1% SDS buffer. Hybridize membrane with reporter gene probe.

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