Team:KIT-Kyoto/Notebook-week5

From 2012.igem.org

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<h2>September 9th</h2>
<h2>September 9th</h2>
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Revision as of 01:22, 24 September 2012






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 GAL440uL
M buffer(TOYOBO)5uL
XbaⅠ(TOYOBO)0.5uL
SpeⅠ(TOYOBO)0.5uL
dH2O4uL
Total50uL


each DNA40uL
3 buffer(NEB)5uL
BglⅡ(NEB)0.5uL
dH2O4.5uL
Total50uL


9/3 GAL423uL
M buffer(TOYOBO)10uL
XbaⅠ(TOYOBO)1uL
SpeⅠ(TOYOBO)1uL
CIP0.5uL
dH2O64.5uL
Total100uL




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

EGFPpSB1C3
DNA template40uL23uL
3 buffer5uL5uL
BglⅡ0.5uL0.5uL
dH2O4.5uL21.5uL
Total50uL50uL




DNA template40uL
4 buffer(NEB)5uL
SpeⅠ0.5uL
100×BSA 0.5uL
CIP0.5uL
dH2O4uL
Total50uL


1uL
1uL
dH2O3uL
Ligation high 2.5uL2.5uL
Total7.5uL




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 template0.25uL
10× KOD plus buffer 5uL5uL
2mM dNTPs5uL
25mM MgSO41.6uL
10P 5’ primer1.5uL
10P 3’ primer1.5uL
KOD plus1uL
dH2O34.15uL
Total50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
58℃30sec30 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
dH2O3uL
Ligation high2.5uL
Total7.5uL


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 template0.25uL
10× KOD plus buffer5uL
2mM dNTPs5uL
25mM MgSO41.6uL
10P 5’ primer1.5uL
10P 3’ primer1.5uL
KOD plus1uL
dH2O34.15uL
Total50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
58℃2min30sec30 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 template1uL
10× KOD plus buffer5uL
2mM dNTPs5uL
25mM MgSO41.6uL
10P 5’ primer1.5uL
10P 3’ primer1.5uL
KOD plus1uL
dH2O33.4uL
Total50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
58℃2min30sec30 cycle
68℃30sec(UAS) or 2min10sec(Except for UAS)30 cycle
68℃30sec(UAS) or 2min10sec(Except for UAS)
14℃




All samples
DNA template1uL
10× KOD plus buffer5uL
2mM dNTPs5uL
25mM MgSO41.6uL
10P 5’ primer1.5uL
10P 3’ primer1.5uL
KOD plus1uL
dH2O33.4uL
Total50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
55℃(-1) or 58℃(-2)2min30sec30 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



UAS40uL
4 buffer(NEB)5uL
EcoRⅠ-HF(NEB)0.5uL
SpeⅠ(NEB0.5uL
100×BSA0.5uL
dH2O3.5uL
Total50uL




All sample
DNA template1uL
10× KOD plus buffer5uL
2mM dNTPs5uL
25mM MgSO41.6uL
10P 5’ primer1.5uL
10P 3’ primer1.5uL
KOD plus1uL
dH2O33.4uL
Total50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
58℃2min30sec30 cycle
68℃2min10sec30 cycle
68℃2min10sec
14℃




1uL
1uL
dH2O3uL
Ligation high2.5uL
Total7.5uL


1uL
dH2O4uL
Ligation high2.5uL
Total7.5uL


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 Act5c40uL
4 buffer(NEB)5uL
XbaⅠ-HF(NEB)0.5uL
BamHⅠ(NEB)0.5uL
100×BSA0.5uL
dH2O3.5uL
Total50uL




G-1 and G-2G-3 and G-4
DNA template1uL4uL
10× KOD plus buffer5uL5uL
2mM dNTPs5uL5uL
25mM MgSO41.6uL1.6uL
10P 5’ primer1uL1uL
10P 3’ primer1uL1uL
KOD plus1uL1uL
dH2O31.4uL
Total50uL50uL


TemperatureTimeCycle
95℃2min
95℃15sec30 cycle
57℃(G-1 and G-3) or 59℃(G-2 and G-4)2min30sec30 cycle
68℃2min10sec30 cycle
68℃2min10sec
14℃




pSB1C3(PCR)40uL
4 buffer(NEB)5uL
EcoRⅠ-HF(NEB)0.5uL
SpeⅠ(NEB)0.5uL
2100×BSA0.5uL
dH2O
Total50uL


13uL
3 buffer(NEB)2uL
BglⅡ(NEB)0.5uL
dH2O2.5uL
Total20uL




GAL420uL
4 buffer(NEB)4uL
XbaⅠ(NEB)0.7uL
SpeⅠ(NEB)0.7uL
100×BSA0.4uL
dH2O14.6uL
Total40uL




GAL440uL
3 buffer(NEB)5uL
BglⅡ(NEB)0.5uL
dH2O4.5uL
Total50uL


GAL4(Bgl Ⅱ cut)40uL
4 buffer(NEB)5uL
SpeⅠ0.5uL
100×BSA0.5uL
dH2O4uL
Total50uL




1uL
2uL
EGFP or LacZ2uL
Ligation high5uL
Total10uL


1uL
2uL
2uL
Ligation high5uL
Total10uL