Team:KIT-Kyoto/Notebook-week4
From 2012.igem.org
(Difference between revisions)
m |
m |
||
Line 97: | Line 97: | ||
<h2>August 27th</h2> | <h2>August 27th</h2> | ||
<br> | <br> | ||
+ | The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately. | ||
+ | <br> | ||
+ | In total 50 pairs were collected. | ||
+ | <br><br> | ||
<h2>August 28th</h2> | <h2>August 28th</h2> | ||
<br> | <br> | ||
Line 102: | Line 106: | ||
<br> | <br> | ||
<h2>August 30th</h2> | <h2>August 30th</h2> | ||
+ | <br> | ||
+ | <strong>Microinjection</strong> | ||
+ | <br> | ||
+ | 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℃. | ||
+ | <br> | ||
+ | 2. NaCl/Triton X-100, 10% Na-hypochloride, H<sub>2</sub>O were kept on ice. | ||
+ | <br> | ||
+ | 3. The tape was placed on the cover glass (3M tape, Cot No. W-18 pastes on the center). | ||
+ | <br> | ||
+ | 4. parafin oil is prepared. | ||
+ | <br> | ||
+ | 5. 1mg/ml DNA in microinjection buffere is prepared. | ||
+ | <br> | ||
+ | 6. glass needles for microinjection were prepared. | ||
+ | <br> | ||
+ | 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 H<sub>2</sub>O. | ||
+ | <br> | ||
+ | 8. The dechorinated embryos were placed on the prepared cover glass then parafin oil was dropped onto the embryos. | ||
+ | <br> | ||
+ | 9. DNA (pUAS-TNFAIP3) was microinjected into embryos. | ||
+ | <br> | ||
+ | 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. | ||
+ | <br> | ||
+ | 11. The plate was kept in the 25℃ incubator. | ||
+ | <br> | ||
+ | In total 692 embryos were microinjected with pUAS-TNFAIP3 DNA (1mg/ml in microinjection buffer). | ||
+ | <br> | ||
+ | Microinjection buffer: 5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8 | ||
+ | <br> | ||
+ | |||
+ | DNA was EtOH-precipitated, washed with 75% EtOH and dissolved to 1 mg/ml in the Microinjection buffer. | ||
+ | <br> | ||
<br> | <br> | ||
<h2>August 31st</h2> | <h2>August 31st</h2> | ||
<br> | <br> | ||
+ | The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. | ||
+ | <br><br> | ||
<h2>September 1st</h2> | <h2>September 1st</h2> | ||
<br> | <br> |
Revision as of 05:51, 22 September 2012
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. August 28thAugust 29thAugust 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. September 1stSeptember 2ndSeptember 3rd |