Team:KIT-Kyoto/Notebook-week6

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Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.
Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.
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<strong>1-2-7 Purification of the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs</strong>
 
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We purified the candidate pSB1C3-UAS-EGFP and pSB1C3-UAS-LacZ DNAs by QIA prep Spin Miniprep Kit from E. coli cultured in LB medium for 16 hours (9/11).
 
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<strong>1-1-47 Digestion of pSB1C3-UAS DNA by Spe1</strong>
 
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We incubated BglⅡ-digested pSB1C3-UAS (9/11) DNA with SpeⅠfor 2 hours. The reaction conditions are shown below.
 
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The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

Revision as of 12:23, 25 September 2012

September 11th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 12th


The transfected cells were examined under the confocal lazer microscope (Olympus, Fv10i)
Results: about 20 % cells were observed to be EGFP-positive, indicating that the P-element plasmid, pUAS-EGFP-TNFAIP3 is functional in Drosophila cells. We therefore decided to microinject this DNA into Drosophila embryos to establish transgenic flies.

The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 13th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 14th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 15th


The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

September 16th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. In total 83 flies from microinjected embryos were mated with yw flies. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.

September 17th


The progeny flies were inspected by dissecting microscope to look for the successfully transformed w+ red eye flies (red eye screening). However, no red eye fly was found.

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