Team:KIT-Kyoto/Notebook-week5

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</td>
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<td width="800px" valign="top"><div id="MIGI">
<td width="800px" valign="top"><div id="MIGI">
<h2>September 4th</h2>
<h2>September 4th</h2>
<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.
<br><br>
<br><br>
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-
<Table Border Cellspacing="0">
 
-
<Tr><Td> 9/3 GAL4 </Td><Td> 40uL </Td></Tr>
 
-
<tr><td> M buffer(TOYOBO) </td><td> 5uL </td></tr>
 
-
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 0.5uL </Td></Tr>
 
-
<tr><td> SpeⅠ(TOYOBO) </td><td> 0.5uL </td></tr><tr>
 
-
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr><tr>
 
-
<td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> each DNA </Td><Td> 40uL </Td></Tr>
 
-
<tr><td> 3 buffer(NEB) </td><td> 5uL </td></tr>
 
-
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr><tr>
 
-
<td> dH<sub>2</sub>O </td><td> 4.5uL </td></tr><tr>
 
-
<td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> 9/3 GAL4 </Td><Td> 23uL </Td></Tr>
 
-
<tr><td> M buffer(TOYOBO) </td><td> 10uL </td></tr>
 
-
<Tr><Td> XbaⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> SpeⅠ(TOYOBO) </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr>
 
-
<td> dH<sub>2</sub>O </td><td> 64.5uL </td></tr>
 
-
<td> Total </td><td> 100uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/b/b0/0904kit.png" width="500" height="300">
 
-
<br><br>
 
<h2>September 5th</h2>
<h2>September 5th</h2>
<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.
<br><br>
<br><br>
-
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </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> BglⅡ </td><td> 0.5uL </td><Td> 0.5uL </Td></tr><tr>
 
-
<td> dH<sub>2</sub>O </td><td> 4.5uL </td><Td> 21.5uL </Td></tr>
 
-
<td> Total </td><td> 50uL </td><Td> 50uL </Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/3/3a/0905akit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> DNA template </Td><Td> 40uL </Td></Tr>
 
-
<tr><td> 4 buffer(NEB) </td><td> 5uL </td></tr>
 
-
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr>
 
-
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr>
 
-
<Tr><Td> CIP </Td><Td> 0.5uL </Td></Tr>
 
-
<td> dH<sub>2</sub>O </td><td> 4uL </td></tr>
 
-
<td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<tr><td></td><td> 1uL </td></tr>
 
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr>
 
-
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr>
 
-
<td> Total </td><td> 7.5uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<img src="http://2012.igem.org/wiki/images/6/63/0905bkit.png" width="500" height="300">
 
-
<br><br>
 
<h2>September 6th</h2>
<h2>September 6th</h2>
<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.
<br><br>
<br><br>
-
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> All samples </Td></Tr>
 
-
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr>
 
-
<tr><td> 10× KOD plus buffer </td><td> 5uL </td></tr>
 
-
<Tr><Td> 2mM dNTPs </Td><Td> 5uL </Td></Tr>
 
-
<Tr><Td> 25mM MgSO4 </Td><Td> 1.6uL </Td></Tr><tr>
 
-
<td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
-
<Tr><Td>  95℃ </Td><Td> 15sec</Td><Td> 30 cycle </Td></Tr>
 
-
<tr><td>  58℃ </td><td> 30sec</td><Td> 30 cycle </Td></tr><tr>
 
-
<td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr>
 
-
<tr><td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr>
 
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/5/53/0906kit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> pSB1C3(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> GAL4(XbaⅠ and SpeⅠ cut) </Td><Td> 1uL </Td></Tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 3uL </td></tr>
 
-
<Tr><Td> Ligation high </Td><Td> 2.5uL </Td></Tr>
 
-
<Tr><td> Total </td><td> 7.5uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 7th</h2>
<h2>September 7th</h2>
<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.
<br><br>
<br><br>
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> All samples </Td></Tr>
 
-
<Tr><Td> DNA template </Td><Td> 0.25uL </Td></Tr>
 
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr>
 
-
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr>
 
-
<Tr><Td> 25mM MgSO4 </Td><Td> 1.6uL </Td></Tr>
 
-
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 34.15uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
-
<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr>
 
-
<tr><td>  58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr>
 
-
<td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr>
 
-
<tr><td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr>
 
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/4/4c/0907akit.png" width="500" height="300">
 
-
<br><br>
 
-
<img src="http://2012.igem.org/wiki/images/6/68/0907bkit.png" width="500" height="300">
 
-
<br><br>
 
-
<img src="http://2012.igem.org/wiki/images/9/9e/0907ckit.png" width="500" height="300">
 
-
<br><br>
 
<h2>September 8th</h2>
<h2>September 8th</h2>
<br>
<br>
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<br><br>
<br><br>
-
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> All samples </Td></Tr>
 
-
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr>
 
-
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr>
 
-
<Tr><Td> 25mM MgSO4 </Td><Td> 1.6uL </Td></Tr>
 
-
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
-
<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr>
 
-
<tr><td>  58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr>
 
-
<td>  68℃ </td></td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr>
 
-
<tr><td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr>
 
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/b/be/0908kit.png" width="500" height="300">
 
-
<br><br>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> All samples </Td></Tr>
 
-
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr>
 
-
<Tr><td> 2mM dNTPs </td><td> 5uL </td></tr>
 
-
<Tr><Td> 25mM MgSO4 </Td><Td> 1.6uL </Td></Tr>
 
-
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
-
<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr>
 
-
<tr><td>  55℃(-1) or 58℃(-2) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr>
 
-
<td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td> 30 cycle </Td></tr>
 
-
<tr><td>  68℃ </td><td> 30sec(UAS) or 2min10sec(Except for UAS) </td><Td></Td></tr>
 
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 9th</h2>
<h2>September 9th</h2>
<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℃.
-
<br><br>
+
<br>
-
TNFAIP3
+
<br>
<br>
Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.
Drosophila S2 cells (2.5 X 10<sup>5</sup> cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.
<br><br>
<br><br>
-
<img src="http://2012.igem.org/wiki/images/1/10/0909akit.png" width="500" height="300">
 
-
<br><br>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> UAS </Td><Td> 40uL </Td></Tr>
 
-
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr>
 
-
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr>
 
-
<Tr><td> SpeⅠ(NEB </td><td> 0.5uL </td></tr>
 
-
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 3.5uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/9/91/0909bkit.png" width="500" height="300">
 
-
<br><br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> All sample </Td></Tr>
 
-
<Tr><Td> DNA template </Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td></Tr>
 
-
<Tr><td> 2mM dNTPs <td> 5uL </td></tr>
 
-
<Tr><Td> 25mM MgSO4 </Td><Td> 1.6uL </Td></Tr>
 
-
<Tr><td> 10P 5’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> 10P 3’ primer </td><td> 1.5uL </td></tr>
 
-
<Tr><td> KOD plus </td><td> 1uL </td></tr>
 
-
<Tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td></tr>
 
-
<Tr><td> Total </td><td> 50uL </td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
 
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
 
-
<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr>
 
-
<tr><td>  58℃ </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr>
 
-
<td>  68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr>
 
-
<tr><td>  68℃ </td><td> 2min10sec </td><Td></Td></tr>
 
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<img src="http://2012.igem.org/wiki/images/b/bb/0909ckit.png" width="500" height="300">
 
-
<br><br>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3uL </Td></Tr>
 
-
<Tr><td> Ligation high </td><td> 2.5uL </td></tr>
 
-
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr>
 
-
</Table>
 
-
<br><BR>
 
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 4uL </Td></Tr>
 
-
<Tr><td> Ligation high </td><td> 2.5uL </td></tr>
 
-
<Tr><Td> Total </Td><Td> 7.5uL </Td></Tr>
 
-
</Table>
 
-
<br><BR>
 
<h2>September 10th</h2>
<h2>September 10th</h2>
<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.
-
<br><br>
 
-
TNFAIP3
 
<br>
<br>
-
At 20 min after removing the medium, 25 uL of serum free medium containing the 1 uL of siLentfect and 200 ng each of pAct5C-GAL4 DNA and pUAS-EGFP-TNFAIP3 DNA was added to the well. At 4 hours after transfection, the transfection medium was removed and the Schneider’s Drosophila medium containing 10% fetal bovine serum was added to each well. The cells were incubated for 48 hours at 25 ℃.
+
<br>
-
<br><br>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> HS or Act5c </Td><Td> 40uL </Td></Tr>
+
-
<Tr><Td> 4 buffer(NEB) </Td><Td> 5uL </Td></Tr>
+
-
<Tr><td> XbaⅠ-HF(NEB) <td> 0.5uL </td></tr>
+
-
<Tr><Td> BamHⅠ(NEB )</Td><Td> 0.5uL </Td></Tr>
+
-
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr>
+
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.5uL </Td></Tr>
+
-
<Tr><Td> Total </Td><Td> 50uL </Td></Tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<img src="http://2012.igem.org/wiki/images/2/27/0910akit.png" width="500" height="300">
+
-
<br><br>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td></Td><Td> G-1 and G-2 </Td><Td> G-3 and G-4 </Td></Tr>
+
-
<tr><td> DNA template </td><td> 1uL </td><Td> 4uL </Td></tr>
+
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 5uL </Td><Td> 5uL </Td></Tr>
+
-
<tr><td> 2mM dNTPs </td><td> 5uL </td><Td> 5uL </Td></tr><tr>
+
-
<td> 25mM MgSO4 </td><td> 1.6uL </td><Td> 1.6uL </Td></tr>
+
-
<tr><td> 10P 5’ primer </td><td> 1uL </td><Td> 1uL </Td></tr>
+
-
<tr><td> 10P 3’ primer </td><td> 1uL </td><Td> 1uL </Td></tr>
+
-
<tr><td> KOD plus </td><td> 1uL </td><Td> 1uL </Td></tr>
+
-
<tr><td> dH<sub>2</sub>O </td><td> 33.4uL </td><Td> 31.4uL </Td></tr>
+
-
<tr><td> Total </td><td> 50uL </td><Td> 50uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> Temperature </Td><Td> Time </Td><Td> Cycle </Td></Tr>
+
-
<tr><td>  95℃ </td><td> 2min </td><Td></Td></tr>
+
-
<Tr><Td>  95℃ </Td><Td> 15sec </Td><Td> 30 cycle </Td></Tr>
+
-
<tr><td>  57℃(G-1 and G-3) or 59℃(G-2 and G-4) </td><td> 2min30sec </td><Td> 30 cycle </Td></tr><tr>
+
-
<td>  68℃ </td><td> 2min10sec </td><Td> 30 cycle </Td></tr>
+
-
<tr><td>  68℃ </td><td> 2min10sec </td><Td></Td></tr>
+
-
<tr><td>  14℃ </td><td> ∞ </td><Td></Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<img src="http://2012.igem.org/wiki/images/e/eb/0910bkit.png" width="500" height="300">
+
-
<br><br>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> pSB1C3(PCR) </Td><Td> 40uL </Td></Tr>
+
-
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr>
+
-
<Tr><Td> EcoRⅠ-HF(NEB) </Td><Td> 0.5uL </Td></Tr>
+
-
<tr><td> SpeⅠ(NEB) </td><Td> 0.5uL </Td></tr><tr>
+
-
<td> 2100×BSA </td><Td> 0.5uL </Td></tr>
+
-
<tr><td> dH<sub>2</sub>O <Td> 3.5uL </Td></tr>
+
-
<tr><td> Total </td><Td> 50uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td></Td><Td> 13uL </Td></Tr>
+
-
<tr><td> 3 buffer(NEB) </td><Td> 2uL </Td></tr>
+
-
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr>
+
-
<tr><td> dH<sub>2</sub>O <Td> 2.5uL </Td></tr>
+
-
<tr><td> Total </td><Td> 20uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<img src="http://2012.igem.org/wiki/images/8/86/0910ckit.png" width="500" height="300">
+
-
<br><br>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> GAL4 </Td><Td> 20uL </Td></Tr>
+
-
<tr><td> 4 buffer(NEB) </td><Td> 4uL </Td></tr>
+
-
<Tr><Td> XbaⅠ(NEB) </Td><Td> 0.7uL </Td></Tr>
+
-
<Tr><Td> SpeⅠ(NEB) </Td><Td> 0.7uL </Td></Tr>
+
-
<Tr><Td> 100×BSA </Td><Td> 0.4uL </Td></Tr>
+
-
<tr><td> dH<sub>2</sub>O </td><Td> 14.6uL </Td></tr>
+
-
<tr><td> Total </td><Td> 40uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<img src="http://2012.igem.org/wiki/images/3/39/0910dkit.png" width="500" height="300">
+
-
<br><br>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> GAL4 </Td><Td> 40uL </Td></Tr>
+
-
<tr><td> 3 buffer(NEB) </td><Td> 5uL </Td></tr>
+
-
<Tr><Td> BglⅡ(NEB) </Td><Td> 0.5uL </Td></Tr>
+
-
<tr><td> dH<sub>2</sub>O </td><Td> 4.5uL </Td></tr>
+
-
<tr><td> Total </td><Td> 50uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> GAL4(Bgl Ⅱ cut) </Td><Td> 40uL </Td></Tr>
+
-
<tr><td> 4 buffer(NEB) </td><Td> 5uL </Td></tr>
+
-
<Tr><Td> SpeⅠ </Td><Td> 0.5uL </Td></Tr>
+
-
<Tr><Td> 100×BSA </Td><Td> 0.5uL </Td></Tr>
+
-
<tr><td> dH<sub>2</sub>O </td><Td> 4uL </Td></tr>
+
-
<tr><td> Total </td><Td> 50uL </Td></tr>
+
-
</Table>
+
-
<br><BR>
+
-
 
+
-
<img src="http://2012.igem.org/wiki/images/6/6d/0910ekit.png" width="500" height="300">
+
-
<br><br>
+
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<tr><td></td><Td> 2uL </Td></tr>
 
-
<Tr><Td> EGFP or LacZ </Td><Td> 2uL </Td></Tr>
 
-
<tr><td> Ligation high </td><Td> 5uL </Td></tr>
 
-
<tr><td> Total </td><Td> 10uL </Td></tr>
 
-
</Table>
 
-
<br><BR>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td></Td><Td> 1uL </Td></Tr>
 
-
<tr><td></td><Td> 2uL </Td></tr>
 
-
<Tr><Td></Td><Td> 2uL </Td></Tr>
 
-
<tr><td> Ligation high </td><Td> 5uL </Td></tr>
 
-
<tr><td> Total </td><Td> 10uL </Td></tr>
 
-
</Table>
 
-
<br><BR>
 
<div align="right"><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6">>>>>>>>>>WEEK6</a></div>
<div align="right"><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6">>>>>>>>>>WEEK6</a></div>
</div>
</div>

Latest revision as of 04:14, 26 September 2012






September 4th


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

September 5th


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

September 6th


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

September 7th


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

September 8th


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℃.

September 9th


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℃.

Drosophila S2 cells (2.5 X 105 cells per well) were plated on the 24 well plate and cultured in the Schneider’s Drosophila medium containing 10% fetal bovine serum at 25 ℃.

September 10th


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.