Xenopus Laevis frogs (Xenopus Express) were anesthetized in Tricaine-S solution (0.5 g/L) and oocytes were extracted by the following sterile surgery protocol: Following a small (~1 cm) incision on the abdomen, the skin and the muscle were cut with scissors. Oocyte sacks were collected gently with forceps and the incision was closed with suture (Oasis PGA Absorbable Suture). The collected oocytes were digested in 0.2 mg/mL collagenase (Sigma) in OR2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) rotating overnight at 18°C. The defolliculated oocytes were washed three times in OR2 and placed in OR2+ solution (OR2 solution supplemented with 1% penicillin/streptomycin and 1.8 mM CaCl2) and kept at 18°C. Healthy oocytes were hand-selected under a microscope and were microinjected with cRNA using a nanoliter injector system (World Precision Instruments). cRNA for this procedure was prepared by mMessage mMachine T7 kit (Thermo Fisher) using wildtype or mutated rabbit TRPV5 DNA as template. Point mutations were generated by Quikchange XL mutagenesis kit (Agilent Genomics).
Nanoliter injector system
Manufactured by World Precision Instruments
The Nanoliter injector system is a precise and versatile instrument designed for the controlled delivery of nanoliter-scale volumes. It provides accurate and repeatable dispensing of small-volume liquids, enabling researchers and scientists to perform a variety of applications requiring high-precision liquid handling.
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2 protocols using nanoliter injector system
1
Xenopus Oocyte Extraction and Injection
2
Xenopus Oocyte Extraction and Injection
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Xenopus Laevis frogs (Xenopus Express) were anesthetized in Tricaine-S solution (0.5 g/L) and oocytes were extracted by the following sterile surgery protocol: Following a small (~1 cm) incision on the abdomen, the skin and the muscle were cut with scissors. Oocyte sacks were collected gently with forceps and the incision was closed with suture (Oasis PGA Absorbable Suture). The collected oocytes were digested in 0.2 mg/mL collagenase (Sigma) in OR2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) rotating overnight at 18°C. The defolliculated oocytes were washed three times in OR2 and placed in OR2+ solution (OR2 solution supplemented with 1% penicillin/streptomycin and 1.8 mM CaCl2) and kept at 18°C. Healthy oocytes were hand-selected under a microscope and were microinjected with cRNA using a nanoliter injector system (World Precision Instruments). cRNA for this procedure was prepared by mMessage mMachine T7 kit (Thermo Fisher) using wildtype or mutated rabbit TRPV5 DNA as template. Point mutations were generated by Quikchange XL mutagenesis kit (Agilent Genomics).
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