Team:KIT-Kyoto/Notebook-week5

From 2012.igem.org

(Difference between revisions)
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<h2>September 4th</h2>
<h2>September 4th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.
Line 103: Line 103:
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<h2>September 5th</h2>
<h2>September 5th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
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-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
-
<Tr><Td></Td><td bgcolor="#f6bfbc">　EGFP　</td><Td>　pSB1C3　</Td></Tr>
+
<Tr><Td></Td><td>　EGFP　</td><Td>　pSB1C3　</Td></Tr>
<tr><td>　DNA template　</td><td>　40uL　</td><Td>　23uL　</Td></tr>
<tr><td>　DNA template　</td><td>　40uL　</td><Td>　23uL　</Td></tr>
<Tr><Td>　3 buffer　</Td><Td>　5uL　</Td><Td>　5uL　</Td></Tr>
<Tr><Td>　3 buffer　</Td><Td>　5uL　</Td><Td>　5uL　</Td></Tr>
Line 183: Line 183:
<h2>September 6th</h2>
<h2>September 6th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
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-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<h2>September 7th</h2>
<h2>September 7th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.
Line 236: Line 236:
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
Line 271: Line 271:
<h2>September 8th</h2>
<h2>September 8th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
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℃.
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℃.
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-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
Line 335: Line 335:
<h2>September 9th</h2>
<h2>September 9th</h2>
<BR>
<BR>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
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℃.
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℃.
Line 341: Line 341:
-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/1/10/0909akit.png" width="500" height="300">
<br><br>
<br><br>
 +
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　UAS　</Td><Td>　40uL　</Td></Tr>
<Tr><Td>　UAS　</Td><Td>　40uL　</Td></Tr>
Line 406: Line 407:
<h2>September 10th</h2>
<h2>September 10th</h2>
<br>
<br>
-
<strong>TNFA and API2</strong>
+
<strong>*TNFA and API2</strong>
<br><br>
<br><br>
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 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.
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-
<strong>Parts</strong>
+
<strong>*Parts</strong>
<br><br>
<br><br>
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br><BR>
<br><BR>
 +
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Cycle　</Td></Tr>
<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Cycle　</Td></Tr>

Revision as of 09:04, 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 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　


Retrieved from "http://2012.igem.org/Team:KIT-Kyoto/Notebook-week5"