Isolation and Purification of DNA Fragments for Microinjection

1. Recombinant DNA containing the reporter construct to be microinjected. Maxiprep DNA purified by CsCl density gradient centrifugation is desirable, although miniprep DNA may also be used.

2. Tris-Borate EDTA buffer (TBE): 89 mM Tris-HCl (pH 8.3), 89 mM boric acid, 2 mM EDTA. Made as a 5X stock solution and diluted to 1X as required with the addition of deionized water (Elga, High Wycombe, Bucks, UK) containing 0.5 pg/mL ethidium bromide (from a 10 mg/mL stock).

3. 0.5-1% (w/v) Agarose (Ultrapure, Bio Rad, Hemel Hempstead, Herts, UK) in 1X TBE/ethidium bromide.

4. 1% (w/v) low-melting-point (LMP) agarose (NuSieve GTG, FMC Bioproducts, Lichfield, Straffs, UK) in 1X TBE/ethidium bromide.

5. ß-Agarase I (1000 U/mL) and 10X Agarase buffer (New England Biolabs, Hitchine, Herts, UK).

6. Phenol (Tris-HCl buffered, pH 8.0, Amresco, Luton, Beds, UK). Store at 4°C.

7. Chloroform.

8. Diethyl ether.

10. Propan-2-ol (isopropanol).

11. Microinjection buffer: 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA. Make up using high-quality, endotoxin-free water that has preferably been embryo-tested (e.g.,W1053, Sigma). Sterilize by passage through a 0.22-|im filter. Store in 10-mL aliquots at -20°C.

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