Team:KIT-Kyoto/Notebook-week5

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September 4th

*TNFA and API2

The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.

*Parts

9/3 GAL4	40uL
M buffer(TOYOBO)	5uL
XbaⅠ(TOYOBO)	0.5uL
SpeⅠ(TOYOBO)	0.5uL
dH₂O	4uL
Total	50uL

each DNA	40uL
3 buffer(NEB)	5uL
BglⅡ(NEB)	0.5uL
dH₂O	4.5uL
Total	50uL

9/3 GAL4	23uL
M buffer(TOYOBO)	10uL
XbaⅠ(TOYOBO)	1uL
SpeⅠ(TOYOBO)	1uL
CIP	0.5uL
dH₂O	64.5uL
Total	100uL

September 5th

*TNFA and API2

The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.

*Parts

	EGFP	pSB1C3
DNA template	40uL	23uL
3 buffer	5uL	5uL
BglⅡ	0.5uL	0.5uL
dH₂O	4.5uL	21.5uL
Total	50uL	50uL

DNA template	40uL
4 buffer(NEB)	5uL
SpeⅠ	0.5uL
100×BSA	0.5uL
CIP	0.5uL
dH₂O	4uL
Total	50uL

	1uL
	1uL
dH₂O	3uL
Ligation high	2.5uL
Total	7.5uL

September 6th

*TNFA and API2

The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.

*Parts

	All samples
DNA template	0.25uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH₂O	34.15uL
Total	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

pSB1C3(XbaⅠ and SpeⅠ cut)	1uL
GAL4(XbaⅠ and SpeⅠ cut)	1uL
dH₂O	3uL
Ligation high	2.5uL
Total	7.5uL

September 7th

*TNFA and API2

The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.

*Parts

	All samples
DNA template	0.25uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH₂O	34.15uL
Total	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

September 8th

*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 eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.

*Parts

	All samples
DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH₂O	33.4uL
Total	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

	All samples
DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH₂O	33.4uL
Total	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
55℃(-1) or 58℃(-2)	2min30sec	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)	30 cycle
68℃	30sec(UAS) or 2min10sec(Except for UAS)
14℃	∞

September 9th

*TNFA and API2

The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.

*Parts

UAS	40uL
4 buffer(NEB)	5uL
EcoRⅠ-HF(NEB)	0.5uL
SpeⅠ(NEB	0.5uL
100×BSA	0.5uL
dH₂O	3.5uL
Total	50uL

	All sample
DNA template	1uL
10× KOD plus buffer	5uL
2mM dNTPs	5uL
25mM MgSO4	1.6uL
10P 5’ primer	1.5uL
10P 3’ primer	1.5uL
KOD plus	1uL
dH₂O	33.4uL
Total	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
58℃	2min30sec	30 cycle
68℃	2min10sec	30 cycle
68℃	2min10sec
14℃	∞

	1uL
	1uL
dH₂O	3uL
Ligation high	2.5uL
Total	7.5uL

	1uL
dH₂O	4uL
Ligation high	2.5uL
Total	7.5uL

September 10th

*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.

*Parts

HS or Act5c	40uL
4 buffer(NEB)	5uL
XbaⅠ-HF(NEB)	0.5uL
BamHⅠ(NEB　)	0.5uL
100×BSA	0.5uL
dH₂O	3.5uL
Total	50uL

	G-1 and G-2	G-3 and G-4
DNA template	1uL	4uL
10× KOD plus buffer	5uL	5uL
2mM dNTPs	5uL	5uL
25mM MgSO4	1.6uL	1.6uL
10P 5’ primer	1uL	1uL
10P 3’ primer	1uL	1uL
KOD plus	1uL	1uL
dH₂O	33.4uL	31.4uL
Total	50uL	50uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	30 cycle
57℃(G-1 and G-3) or 59℃(G-2 and G-4)	2min30sec	30 cycle
68℃	2min10sec	30 cycle
68℃	2min10sec
14℃	∞

pSB1C3(PCR)	40uL
4 buffer(NEB)	5uL
EcoRⅠ-HF(NEB)	0.5uL
SpeⅠ(NEB)	0.5uL
2100×BSA	0.5uL
dH₂O	3.5uL
Total	50uL

	13uL
3 buffer(NEB)	2uL
BglⅡ(NEB)	0.5uL
dH₂O	2.5uL
Total	20uL

GAL4	20uL
4 buffer(NEB)	4uL
XbaⅠ(NEB)	0.7uL
SpeⅠ(NEB)	0.7uL
100×BSA	0.4uL
dH₂O	14.6uL
Total	40uL

GAL4	40uL
3 buffer(NEB)	5uL
BglⅡ(NEB)	0.5uL
dH₂O	4.5uL
Total	50uL

GAL4(Bgl Ⅱ cut)	40uL
4 buffer(NEB)	5uL
SpeⅠ	0.5uL
100×BSA	0.5uL
dH₂O	4uL
Total	50uL

	1uL
	2uL
EGFP or LacZ	2uL
Ligation high	5uL
Total	10uL

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

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