Methods

1. Restrain the animal with one hand (see Chapter 14) and use a sharp pair of fine scissors to cut off approx 1 cm of tail (see Note 1). Place this tissue in 700 pL of PK buffer contained in an autoclaved 1.5-mL Eppendorf-type centrifuge tube. At the same time, tag the animal (see Note 2).

2. Mince the tail with a fine pair of scissors (this step is optional; see Note 3).

3. Incubate at 55°C overnight, preferably with gentle agitation

4. Add 5 pL of 1 mg/mL RNase A. Incubate at 37°C for 1-2 h.

5. Add 700 jjL of Tris buffered phenol. Extract the digested tail by gentle mixing with the phenol until homogeneous.

6 Separate the phases by centrifugation at 10,000 rpm in a microcentrifuge for 5 min. Transfer the viscous upper aqueous phase and the interphase to a fresh tube.

7 Add 700 pL of Tris buffered phenol/chloroform/isoamylalcohol (24:24:1).

Viewed from below

Fig. 1. A scheme for marking mice by toe clipping. The toe is clipped up to the first joint. Up to 100 animals can be so marked. See discussion on p. 312.

Gently mix the phases until emulsified and separate by centrifugation at 10,000 rpm in a microcentrifuge for 5 min.

8. Carefully avoiding the interphase, transfer the aqueous phase to a fresh tube. Add approx 1 mL 100% ethanol. Gently mix. A stringy white precipitate of genomic DNA should be formed.

9. Pellet the precipitate by centrifugation at 10,000 rpm in a microcentrifuge for 2 min.

10. Discard the supernatant and rinse the pellet with 70% (v/v) ethanol. It is permissible to vortex gently at this stage. Repellet the DNA by centrifugation for 1 min at 10,000 rpm in a microcentrifuge.

11. Remove as much of the supernatant as possible and dry the pellet under vacuum.

12. Resuspend the pellet in 250 |oL of sterile distilled water. Incubate at 37°C for several hours to enable the DNA to dissolve.

13. Assay the yield of DNA by UV spectrophotometry (260 nm) (see Note 4).

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