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cDNA was prepared from 2 ug of poly A+ RNA (equal parts from stage 10.5 and stage 11.5 gastrulae). EcoRI-XhoI cut cDNA was then ligated into UniZap-XR (Stratagene) with EcoRI at the 5' end and XhoI at the 3' end. The library was mass excised and used to infect Top10F'. Clones were picked into freezing medium (per liter 15 g tryptone, 10g yeast extract, 5g NaCl, 36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM Na-citrate, 0.4 mM MgSO4 7 H2O, 6.8 mM (NH4)2SO4, 4 % w/v glycerol) and grown for 24 hours. Original library construction by Bruce Blumberg (Cho et al 1991 Cell 67, 1111-1120).
Nucleotide
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