Team:KIT-Kyoto/Notebook-week6

September 11th

*TNFA and API2

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

*Parts

pSB1C3-UAS-1, -2, -3, -4	5uL
3buffer(NEB)	2uL
BglⅡ	0.5uL
dH2O	12.5uL
Total	20uL

GAL4	40uL
4 buffer(NEB)	5uL
XbaⅠ(NEB)	0.5uL
SpeⅠ(NEB)	0.5uL
CIP	0.5uL
100×BSA	0.5uL
dH2O	3uL
Total	40uL

	1uL
	2.5uL
	2.5uL
Ligation high	6uL
Total	12uL

	2uL
	2.5uL
	1.5uL
Ligation high	6uL
Total	12uL

September 12th

*TNFA and API2

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

*Parts

pSB1C3-UAS-1, -2	40uL
3buffer(NEB)	5uL
SpeⅠ	1uL
100×BSA	0.5uL
dH2O	3.5uL
Total	50uL

	1uL
	2uL
EGFP or LacZ	2uL
Ligation high	2.5uL
Total	7.5uL

	2uL
	2uL
	2uL
Ligation high	6uL
Total	12uL

September 13th

*TNFA and API2

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

*Parts

DNA sample	1uL
4 buffer(NEB)	0.5uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.05uL
dH2O	3.25uL
Total	5uL

DNA sample	1uL
3 buffer(NEB)	0.5uL
BglⅡ	0.2uL
dH2O	3.3uL
Total	5uL

	1uL
	1.5uL
dH2O	2.5uL
Ligation high	2.5uL
Total	7.5uL

	2.5uL
	2.5uL
	2uL
Ligation high	7uL
Total	14uL

	1uL
	2uL
dH2O	2uL
Ligation high	5uL
Total	10uL

September 14th

*TNFA and API2

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℃.

*Parts

DNA sample	1uL
4 buffer(NEB)	1uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.1uL
dH2O	7.5uL
Total	10uL

	2uL
	3uL
	1uL(HS) or 2.5uL(Act5c)
Ligation high	6uL(HS) or 7.5uL(Act5c)
Total	12uL(HS) or 15uL(Act5c)

September 15th

*TNFA and API2

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

*Parts

DNA sample	1uL
4 buffer(NEB)	1uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.1uL
dH2O	7.5uL
Total	10uL

September 16th

*TNFA and API2

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

*TNFA and API2

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.

*Parts

DNA sample	1uL
4 buffer(NEB)	1uL
EcoRⅠ-HF(NEB)	0.2uL
SpeⅠ(NEB)	0.2uL
100×BSA	0.1uL
dH2O	7.5uL
Total	10uL