Team:KIT-Kyoto/Notebook-week4

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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 high	2.5uL
dH₂O	5uL
Total	7.5uL

August 28th

TNFA and API2

August 29th

Parts

pEGFP-C2-1, -2, -3, -4	1uL
10×H buffer(TOYOBO)	0.5uL
XhoⅠ	0.1uL
PstⅠ	0.1uL
dH₂O	3.3uL
Total	5uL

	All samples
DNA template	0.5uL
10× KOD plus buffer 5uL	10uL
2mM dNTPs	10uL
25mM MgSO4	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH₂O	68.3uL
Total	100uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	25 cycle
58℃	30sec	25 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 template	0.5uL
10× KOD plus buffer 5uL	10uL
2mM dNTPs	10uL
25mM MgSO4	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH₂O	68.3uL
Total	100uL

Temperature	Time	Cycle
95℃	2min
95℃	15sec	25 cycle
58℃	30sec	25 cycle
68℃	30sec	25 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 template	0.3uL
10× KOD plus buffer	10uL
2mM dNTPs	10uL
25mM MgSO4	3.2uL
10P 5’ primer	3uL
10P 3’ primer	3uL
KOD plus	2uL
dH₂O	68.5uL
Total	100uL

Tempreture	Time	Circle
95℃	2min
95℃	15sec	25 cycle
59℃	30sec	25 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 template	0.2uL
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	34.2uL
Total	50uL

Tempreture	Time	Circle
95℃	2min
95℃	15sec	25 cycle
55℃(GAL4) and 58℃(Except for GAL4)	30sec	25 cycle
68℃	2min30sec	25 cycle
68℃	2min30sec
14℃	∞

@@ Line 105: / Line 105: @@
 <strong>Parts</strong>
 <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="https://static.igem.org/mediawiki/2012/e/e6/0826bkit.png" width="500" height="300">
 <br><br>
@@ Line 117: / Line 124: @@
 <strong>Parts</strong>
 <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="https://static.igem.org/mediawiki/2012/9/97/0828kit.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>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="https://static.igem.org/mediawiki/2012/d/d2/0829b.png" width="500" height="300">
 <br><br>
@@ Line 164: / Line 203: @@
 <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>