Transgenic Diagnosis by the Polymerase Chain Reaction PCR

1. PK digestion buffer (10X stock): 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 1% (v/v) Tween-20. Store at room temperature. Dilute to 1X with distilled water.

2. Proteinase K (Boehringer): 10 mg/mL (100X) stock solution in 50 mM Tris-HCl (pH 8.0), 1 mM CaCl2. Filter-sterilize, and store in aliquots at 4°C.

3. Taq polymerase: Amplitaq (5 U/||L) and 10X PCR buffer II (Perkin Elmer, Warrington, Cheshire, UK).

4. MgCl2 solution: 10X stock for PCR, depending on optimal Mg2+ concentration for primers being used (e.g., 0.5-2.5 mM final). Store at -20°C in 1-mL aliquots.

5. dNTP mix: 100 mM solutions (Ultrapure, Pharmacia, Little Chalfont, Bucks, UK). Combine and dilute to 2 mM each with TE (pH 7.5) to give a 10X dNTP stock. Store in aliquots at -20°C.

6. PCR primers: Suitable transgene-specific and internal control primers. Store at 10X concentration (2 |M, 2 pmol/|L) in distilled water at -20°C.

8. 1.5% (w/v) Agarose (Ultrapure, Bio Rad) in 1X TBE/ethidium bromide.

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