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Team:KIT-Kyoto/Notebook-week4
From 2012.igem.org
August 27thThe female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately. In total 50 pairs were collected. TNFA and API2 Parts
August 28thTNFA and API2 Parts August 29thParts
August 30thMicroinjection 1. In the morning, w; Δ2,3 (female-virgin) were crossed with yw (male) in the cage and kept for 3 to 4 hours at 25℃. 2. NaCl/Triton X-100, 10% Na-hypochloride, H2O were kept on ice. 3. The tape was placed on the cover glass (3M tape, Cot No. W-18 pastes on the center). 4. parafin oil is prepared. 5. 1mg/ml DNA in microinjection buffere is prepared. 6. glass needles for microinjection were prepared. 7. The early embryos were collected on the nylon mesh and washed 3-4 times with NaCl/Triton X-100. Then embryos were dechorinated with 5 % Na-hypochloride. Then the dechorionated embryos were washed 7-8 times with H2O. 8. The dechorinated embryos were placed on the prepared cover glass then parafin oil was dropped onto the embryos. 9. DNA (pUAS-TNFAIP3) was microinjected into embryos. 10. After microinjection, the injected embryos were removed with the tape on the cover glass and placed onto the new egg plate. The tapes keep upside down from avoiding dry the embryos. 11. The plate was kept in the 25℃ incubator. In total 692 embryos were microinjected with pUAS-TNFAIP3 DNA (1mg/ml in microinjection buffer). Microinjection buffer: 5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8 DNA was EtOH-precipitated, washed with 75% EtOH and dissolved to 1 mg/ml in the Microinjection buffer. 
August 31stThe hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. Parts
September 1stTNFA and API2 The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. About 20 larvae were put into a fly food vial. Parts 
September 2ndTNFA and API2 The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. In total 144 larvae were collected. The hatching efficiency after microinjection was calculated to be 20.8%. September 3rdTNFA and API2 Parts
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