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.
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.
Was this article helpful?