Solutions 500mM K4Fe(CN)6.3H2O (Potassium Ferrocyanide) (Sigma: P9387) in DDW 500mM K3Fe(CN)6 (Potassium Ferricyanide) in DDW Solution A : 0.2M NaH2PO4.H2O (27.6g/l) Solution B : 0.2M Na2HPO4 (28,4g/l) Phosphate buffer : 23ml of 0.2M NaH2PO4.H2O, 77ml of 0.2M Na2HPO4 and add 100ml DDW. the pH will be approx 7.3 0.5M EGTA in 0.1M Phosphate buffer, pH increase to 7. FW 380.4, so 19.02g for making 100ml of 0.5M EGTA. Ethylene Glycol-bis (b-aminoethyl ether)N,N,N',N'-tetraacetic acid) (Sigma E-4378) Wash buffer : 200ml of wash buffer combine by 23ml of 0.2M NaH2PO4.H2O, 77ml of 0.2M Na2HPO4, 400ml of 1M MgCl2 2ml of 0.5M EGTA 0.2ml of 20% NP-40 0.2ml of 10% Na Deoxycholate DDW up to 200ml Fixing buffer : 198ml 0.1M phosphate buffer, 400ml of 1M MgCl2 2ml of 0.5M EGTA X-gal : 40 mg/ml in diemthylformamide, covered by foil and store at -20C. 10% formalin (20L) 95.36g KH2PO4 192g K2HPO4 200 ml 40% formadhyde the pH should be 7.4 Fixtive using fix buffer to make 0.2% Glutaraldehyde fixative, eg: add 0.2ml 25% Glutaraldehyde (EM grade) into 24.8ml fixing buffer. (fresh make every time) For E10 - E12 embryo - fix the embryo on ice for 10 minutes (with at least 20ml fixative); Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au David Bowtell PMCI October 1998 |
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