Transgenic Diagnosis by PCR

Presented here is a simple and quick method for the identification of trans-genic animals or embryos by PCR from small tissue samples. The starting sample can be a small (0.5 cm) tail biopsy for animals or a portion of ex-traembryonic tissue (placenta or extraembryonic membranes) for embryos (see Note 29).

1. Boil the tissue sample to be analyzed for 5 min in 0.5 mL of proteinase K digestion buffer (see Note 30).

2. Add 5 ||L of proteinase K solution (final concentration 0.1 mg/mL), mix well by inversion, and digest the tissue samples overnight at 55°C.

3. Vortex the samples briefly to disperse any clumps of partially digested tissue. Reboil the tissue sample for 5 min (see Note 30).

4. Spin at 15,000g for 20 min (4°C) to pellet any cellular debris. Use 1 |L as a template for subsequent PCR diagnosis.

5. Set up a 20-|L PCR reaction containing; 1X PCR buffer, 1X dNTP mix, 1X MgCl2, 1X transgene-specific primers (0.2 ||M each), control primers (0.2 |M each), template, and 1.5 U of Taq polymerase. Overlay with mineral oil, and amplify using suitable conditions in a thermal cycler. It is useful to include both positive and negative controls (see Notes 31 and 32).

6. Run out the reaction products on an agarose-ethidium bromide gel. Positive samples should show the presence of both the transgene-specific and control bands.

Was this article helpful?

0 0

Post a comment