Team:KIT-Kyoto/Notebook-week4

August 27th

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.
In total 50 pairs were collected.

Large scale purification of the pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) for microinjection into Drosophila embryos.
To purify pUAS-flag-TNFAIP3 DNA, E. coli carrying the plasmid was cultured in 100 mL of LB ampicillin(+) nedium at 37℃ for 16 hours.

August 28th

1.Sequencing of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA
To determine nucleotide sequence of pUAS-flag-TNFAIP3 (pTFW-TNFAIP3) and pTGW-TNFAIP3 DNA, we send the DNAs to the company. The sequencing data confirmed that pUAS-flag-TNFAIP3 is properly constructed.

2. Midi-prep purification of the pUAS-flag-TNFAIP3 DNA
The pUAS-flag-TNFAIP3 DNA was purified from E. coli cultured on on August 22　according to protocol using Pure LinkTM HiPure Plasmid Midiprep. Concentration of the obtained DNA was measured and adjusted it to be 1mg/mL

August 30th

Micro-injection of the pUAS-flag-TNFAIP3 DNA into Drosophila embryos
Microinjection
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-flag-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-flag-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 31st

The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.

September 1st

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.

September 2nd

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%.