Team:KIT-Kyoto/Notebook-week4

From 2012.igem.org

(Difference between revisions)
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<strong>Parts</strong>
<strong>Parts</strong>
<br><br>
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>pEGFP-C2(cut)</Td><Td>1uL</Td></Tr>
 +
<tr><td>Ligation high</td><td>2.5uL</td></tr><tr>
 +
<td>dH<sub>2</sub>O</td><td>5uL</td></tr><tr>
 +
<td>Total<td>7.5uL</td></tr>
 +
</Table>
 +
<br><BR>
<img src="http://2012.igem.org/wiki/images/e/e6/0826bkit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/e/e6/0826bkit.png" width="500" height="300">
<br><br>
<br><br>
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<strong>Parts</strong>
<strong>Parts</strong>
<br><br>
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>pEGFP-C2-1, -2, -3, -4</Td><Td>1uL</Td></Tr>
 +
<tr><td>10×H buffer(TOYOBO)</td><td>0.5uL</td></tr><tr>
 +
<tr><td>XhoⅠ</td><td>0.1uL</td></tr><tr>
 +
<tr><td>PstⅠ</td><td>0.1uL</td></tr><tr>
 +
<td>dH<sub>2</sub>O</td><td>3.3uL</td></tr><tr>
 +
<td>Total<td>5uL</td></tr>
 +
</Table>
 +
<br><BR>
<img src="http://2012.igem.org/wiki/images/9/97/0828kit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/9/97/0828kit.png" width="500" height="300">
<br><br>
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td></Td><Td>All samples</Td></Tr>
 +
<Tr><Td>DNA template</Td><Td>0.5uL</Td></Tr>
 +
<tr><td>10× KOD plus buffer 5uL</td><td>10uL</td></tr>
 +
<Tr><Td>2mM dNTPs</Td><Td>10uL</Td></Tr>
 +
<Tr><Td>25mM MgSO4</Td><Td>3.2uL</Td></Tr>
 +
<td>10P 5’ primer</td><td>3uL</td></tr>
 +
<Tr><td>10P 3’ primer</td><td>3uL</td></tr>
 +
<Tr><td>KOD plus</td><td>2uL</td></tr>
 +
<Tr><td>dH<sub>2</sub>O</td><td>68.3uL</td></tr>
 +
<Tr><td>Total</td><td>100uL</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>25 cycle</Td></Tr>
 +
<tr><td>58℃</td><td>30sec</td><Td>25 cycle</Td></tr><tr>
 +
<td>68℃</td><td>30sec(UAS) or 1min(EGFP))</td><Td>25 cycle</Td></tr>
 +
<tr><td>68℃</td><td>30sec(UAS) or 1min(EGFP)</td><Td></Td></tr>
 +
<tr><td>14℃</td><td>∞</td><Td></Td></tr>
 +
</Table>
 +
<br><BR>
<img src="http://2012.igem.org/wiki/images/d/d2/0829b.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/d/d2/0829b.png" width="500" height="300">
<br><br>
<br><br>
Line 164: Line 203:
<br><br>
<br><br>
 +
 +
<strong>Parts</strong>
 +
<br><br>
 +
<Table Border Cellspacing="0">
 +
<Tr><Td></Td><Td>All samples</Td></Tr>
 +
<Tr><Td>DNA template</Td><Td>0.5uL</Td></Tr>
 +
<tr><td>10× KOD plus buffer 5uL</td><td>10uL</td></tr>
 +
<Tr><Td>2mM dNTPs</Td><Td>10uL</Td></Tr>
 +
<Tr><Td>25mM MgSO4</Td><Td>3.2uL</Td></Tr>
 +
<td>10P 5’ primer</td><td>3uL</td></tr>
 +
<Tr><td>10P 3’ primer</td><td>3uL</td></tr>
 +
<Tr><td>KOD plus</td><td>2uL</td></tr>
 +
<Tr><td>dH<sub>2</sub>O</td><td>68.3uL</td></tr>
 +
<Tr><td>Total</td><td>100uL</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>25 cycle</Td></Tr>
 +
<tr><td>58℃</td><td>30sec</td><Td>25 cycle</Td></tr><tr>
 +
<td>68℃</td><td>30sec</td><Td>25 cycle</Td></tr>
 +
<tr><td>68℃</td><td>30sec</td><Td></Td></tr>
 +
<tr><td>14℃</td><td>∞</td><Td></Td></tr>
 +
</Table>
 +
<br><BR>

Revision as of 11:34, 23 September 2012






August 27th


The female-virgin flies (w; Δ2,3) and male flies (yw) were collected for microinjection. The collected flies were kept in the 25℃ incubator separately.
In total 50 pairs were collected.

TNFA and API2

Parts

pEGFP-C2(cut)1uL
Ligation high2.5uL
dH2O5uL
Total7.5uL






August 28th


TNFA and API2

August 29th


Parts

pEGFP-C2-1, -2, -3, -41uL
10×H buffer(TOYOBO)0.5uL
XhoⅠ0.1uL
PstⅠ0.1uL
dH2O3.3uL
Total5uL




All samples
DNA template0.5uL
10× KOD plus buffer 5uL10uL
2mM dNTPs10uL
25mM MgSO43.2uL
10P 5’ primer3uL
10P 3’ primer3uL
KOD plus2uL
dH2O68.3uL
Total100uL


TemperatureTimeCycle
95℃2min
95℃15sec25 cycle
58℃30sec25 cycle
68℃30sec(UAS) or 1min(EGFP))25 cycle
68℃30sec(UAS) or 1min(EGFP)
14℃




August 30th


Microinjection
1. In the morning, w; Δ2,3 (female-virgin) were crossed with yw (male) in the cage and kept for 3 to 4 hours at 25℃.
2. NaCl/Triton X-100, 10% Na-hypochloride, H2O were kept on ice.
3. The tape was placed on the cover glass (3M tape, Cot No. W-18 pastes on the center).
4. parafin oil is prepared.
5. 1mg/ml DNA in microinjection buffere is prepared.
6. glass needles for microinjection were prepared.
7. The early embryos were collected on the nylon mesh and washed 3-4 times with NaCl/Triton X-100. Then embryos were dechorinated with 5 % Na-hypochloride. Then the dechorionated embryos were washed 7-8 times with H2O.
8. The dechorinated embryos were placed on the prepared cover glass then parafin oil was dropped onto the embryos.
9. DNA (pUAS-TNFAIP3) was microinjected into embryos.
10. After microinjection, the injected embryos were removed with the tape on the cover glass and placed onto the new egg plate. The tapes keep upside down from avoiding dry the embryos.
11. The plate was kept in the 25℃ incubator.
In total 692 embryos were microinjected with pUAS-TNFAIP3 DNA (1mg/ml in microinjection buffer).
Microinjection buffer: 5 mM KCl, 0.1 mM Sodium Phosphate, pH6.8
DNA was EtOH-precipitated, washed with 75% EtOH and dissolved to 1 mg/ml in the Microinjection buffer.



August 31st


The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.

Parts

All samples
DNA template0.5uL
10× KOD plus buffer 5uL10uL
2mM dNTPs10uL
25mM MgSO43.2uL
10P 5’ primer3uL
10P 3’ primer3uL
KOD plus2uL
dH2O68.3uL
Total100uL


TemperatureTimeCycle
95℃2min
95℃15sec25 cycle
58℃30sec25 cycle
68℃30sec25 cycle
68℃30sec
14℃


September 1st


The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. About 20 larvae were put into a fly food vial.

Parts



All samples
DNA template0.3uL
10× KOD plus buffer10uL
2mM dNTPs10uL
25mM MgSO43.2uL
10P 5’ primer3uL
10P 3’ primer3uL
KOD plus2uL
dH2O68.5uL
Total100uL


TempretureTimeCircle
95℃2min
95℃15sec25 cycle
59℃30sec25 cycle
68℃1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)25 cycle
68℃1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)
14℃




September 2nd


The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator. In total 144 larvae were collected. The hatching efficiency after microinjection was calculated to be 20.8%.

September 3rd


Parts

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


TempretureTimeCircle
95℃2min
95℃15sec25 cycle
55℃(GAL4) and 58℃(Except for GAL4)30sec25 cycle
68℃2min30sec25 cycle
68℃2min30sec
14℃