Team:KIT-Kyoto/Notebook-week4

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(Difference between revisions)

Revision as of 01:59, 24 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

Parts

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	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	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

TNFA and API2

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

Temperature	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

TNFA and API2

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

TNFA and API2

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	1u　L
dH₂O	34.2uL
Total	50uL

Temperature	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 106: / Line 106: @@
 <br><br>
 <Table Border Cellspacing="0">
-<Tr><Td>pEGFP-C2(cut)</Td><Td>1uL</Td></Tr>
+<Tr><Td>　pEGFP-C2(cut)　</Td><Td>　1uL　</Td></Tr>
-<tr><td>Ligation high</td><td>2.5uL</td></tr><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>　dH<sub>2</sub>O　</td><td>　5uL　</td></tr><tr>
-<td>Total<td>7.5uL</td></tr>
+<td>　Total　<td>　7.5uL　</td></tr>
 </Table>
 <br><BR>
@@ Line 127: / Line 127: @@
 <br><br>
 <Table Border Cellspacing="0">
-<Tr><Td>pEGFP-C2-1, -2, -3, -4</Td><Td>1uL</Td></Tr>
+<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>　10×H buffer(TOYOBO)　</td><td>　0.5uL　</td></tr><tr>
-<tr><td>XhoⅠ</td><td>0.1uL</td></tr><tr>
+<tr><td>　XhoⅠ　</td><td>　0.1uL　</td></tr><tr>
-<tr><td>PstⅠ</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>　dH<sub>2</sub>O　</td><td>　3.3uL　</td></tr><tr>
-<td>Total<td>5uL</td></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></Td><Td>　All samples　</Td></Tr>
-<Tr><Td>DNA template</Td><Td>0.5uL</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>　10× KOD plus buffer　</td><td>　10uL　</td></tr>
-<Tr><Td>2mM dNTPs</Td><Td>10uL</Td></Tr>
+<Tr><Td>　2mM dNTPs　</Td><Td>　10uL　</Td></Tr>
-<Tr><Td>25mM MgSO4</Td><Td>3.2uL</Td></Tr>
+<Tr><Td>　25mM MgSO4　</Td><Td>　3.2uL　</Td></Tr>
-<td>10P 5’ primer</td><td>3uL</td></tr>
+<td>　10P 5’ primer　</td><td>　3uL　</td></tr>
-<Tr><td>10P 3’ 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>　KOD plus　</td><td>　2uL　</td></tr>
-<Tr><td>dH<sub>2</sub>O</td><td>68.3uL</td></tr>
+<Tr><td>　dH<sub>2</sub>O　</td><td>　68.3uL　</td></tr>
-<Tr><td>Total</td><td>100uL</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>　Temperature　</Td><Td>　Time　</Td><Td>　Cycle　</Td></Tr>
-<tr><td>95℃</td><td>2min</td><Td></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>　95℃　</Td><Td>　15sec　</Td><Td>　25 cycle　</Td></Tr>
-<tr><td>58℃</td><td>30sec</td><Td>25 cycle</Td></tr><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>
+<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>　68℃　</td><td>　30sec(UAS) or 1min(EGFP)　</td><Td></Td></tr>
-<tr><td>14℃</td><td>∞</td><Td></Td></tr>
+<tr><td>　14℃　</td><td>　∞　</td><Td></Td></tr>
 </Table>
 <br><BR>
@@ Line 209: / Line 211: @@
 <br><br>
 <Table Border Cellspacing="0">
-<Tr><Td></Td><Td>All samples</Td></Tr>
+<Tr><Td></Td><Td>　All samples　</Td></Tr>
-<Tr><Td>DNA template</Td><Td>0.5uL</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>　10× KOD plus buffer　</td><td>　10uL　</td></tr>
-<Tr><Td>2mM dNTPs</Td><Td>10uL</Td></Tr>
+<Tr><Td>　2mM dNTPs　</Td><Td>　10uL　</Td></Tr>
-<Tr><Td>25mM MgSO4</Td><Td>3.2uL</Td></Tr>
+<Tr><Td>　25mM MgSO4　</Td><Td>　3.2uL　</Td></Tr>
-<td>10P 5’ primer</td><td>3uL</td></tr>
+<td>　10P 5’ primer　</td><td>　3uL　</td></tr>
-<Tr><td>10P 3’ 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>　KOD plus　</td><td>　2uL　</td></tr>
-<Tr><td>dH<sub>2</sub>O</td><td>68.3uL</td></tr>
+<Tr><td>　dH<sub>2</sub>O　</td><td>　68.3uL　</td></tr>
-<Tr><td>Total</td><td>100uL</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>　Temperature</Td><Td>　Time　</Td><Td>Cycle</Td></Tr>
-<tr><td>95℃</td><td>2min</td><Td></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>　　95℃　</Td><Td>　15sec　</Td><Td>　25 cycle　</Td></Tr>
-<tr><td>58℃</td><td>30sec</td><Td>25 cycle</Td></tr><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>
+<td>　　68℃　</td><td>　30sec　</td><Td>　25 cycle　</Td></tr>
-<tr><td>68℃</td><td>30sec</td><Td></Td></tr>
+<tr><td>　　68℃　</td><td>　30sec　</td><Td></Td></tr>
-<tr><td>14℃</td><td>∞</td><Td></Td></tr>
+<tr><td>　　14℃　</td><td>　∞　</td><Td></Td></tr>
 </Table>
 <br><BR>
@@ Line 246: / Line 249: @@
 <br><br>
 <Table Border Cellspacing="0">
-<Tr><Td></Td><Td>All samples</Td></Tr>
+<Tr><Td></Td><Td>　All samples　</Td></Tr>
-<tr><td>DNA template</td><td>0.3uL</td></tr>
+<tr><td>　DNA template　</td><td>　0.3uL　</td></tr>
-<Tr><Td>10× KOD plus buffer</Td><Td>10uL</Td></Tr>
+<Tr><Td>　10× KOD plus buffer　</Td><Td>　10uL　</Td></Tr>
-<tr><td>2mM dNTPs</td><td>10uL</td></tr><tr>
+<tr><td>　2mM dNTPs　</td><td>　10uL　</td></tr><tr>
-<td>25mM MgSO4</td><td>3.2uL</td></tr>
+<td>　25mM MgSO4　</td><td>　3.2uL　</td></tr>
-<td>10P 5’ primer</td><td>3uL</td></tr>
+<td>　10P 5’ primer　</td><td>　3uL　</td></tr>
-<td>10P 3’ primer</td><td>3uL</td></tr>
+<td>　10P 3’ primer　</td><td>　3uL　</td></tr>
-<td>KOD plus</td><td>2uL</td></tr>
+<td>　KOD plus　</td><td>　2uL　</td></tr>
-<td>dH<sub>2</sub>O</td><td>68.5uL</td></tr>
+<td>　dH<sub>2</sub>O　</td><td>　68.5uL　</td></tr>
-<td>Total<td>100uL</td></tr>
+<td>　Total　<td>　100uL　</td></tr>
 </Table>
 <br><BR>
 <Table Border Cellspacing="0">
-<Tr><Td>Tempreture</Td><Td>Time</Td><Td>Circle</Td></Tr>
+<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
-<tr><td>95℃</td><td>2min</td><Td></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>　　95℃　</Td><Td>　15sec　</Td><Td>　25 cycle　</Td></Tr>
-<tr><td>59℃</td><td>30sec</td><Td>25 cycle</Td></tr><tr>
+<tr><td>　　59℃　</td><td>　30sec　</td><Td>　25 cycle　</Td></tr><tr>
-<td>68℃</td><td>1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)</td><Td>25 cycle</Td></tr>
+<td>　　68℃　</td><td>　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　</td><Td>　25 cycle　</Td></tr><tr>
-<td>68℃</td><td>1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)</td><Td></Td></tr>
+<td>　　68℃　</td><td>　1min30sec(Act5c and LacZ) or 2min(GAL4 and U-T)　</td><Td></Td></tr><tr>
-<td>14℃</td><td>∞</td><Td></Td></tr>
+<td>　　14℃　</td><td>　∞　</td><Td></Td></tr>
 </Table>
 <br><br>
@@ Line 287: / Line 290: @@
 <br><br>
 <Table Border Cellspacing="0">
-<Tr><Td></Td><Td>All samples</Td></Tr>
+<Tr><Td></Td><Td>　All samples　</Td></Tr>
-<tr><td>DNA template</td><td>0.2uL</td></tr>
+<tr><td>　DNA template　</td><td>　0.2uL　</td></tr>
-<Tr><Td>10× KOD plus buffer</Td><Td>5uL</Td></Tr>
+<Tr><Td>　10× KOD plus buffer　</Td><Td>　5uL　</Td></Tr>
-<tr><td>2mM dNTPs</td><td>5uL</td></tr><tr>
+<tr><td>　2mM dNTPs　</td><td>　5uL　</td></tr><tr>
-<td>25mM MgSO4</td><td>1.6uL</td></tr>
+<td>　25mM MgSO4　</td><td>　1.6uL　</td></tr><tr>
-<td>10P 5’ primer</td><td>1.5uL</td></tr>
+<td>　10P 5’ primer　</td><td>　1.5uL　</td></tr><tr>
-<td>10P 3’ primer</td><td>1.5uL</td></tr>
+<td>　10P 3’ primer　</td><td>　1.5uL　</td></tr><tr>
-<td>KOD plus</td><td>1uL</td></tr>
+<td>　KOD plus　</td><td>　1u　L</td></tr><tr>
-<td>dH<sub>2</sub>O</td><td>34.2uL</td></tr>
+<td>　dH<sub>2</sub>O　</td><td>　34.2uL　</td></tr><tr>
-<td>Total<td>50uL</td></tr>
+<td>　Total　<td>　50uL　</td></tr>
 </Table>
 <br><BR>
 <Table Border Cellspacing="0">
-<Tr><Td>Tempreture</Td><Td>Time</Td><Td>Circle</Td></Tr>
+<Tr><Td>　Temperature　</Td><Td>　Time　</Td><Td>　Circle　</Td></Tr>
-<tr><td>95℃</td><td>2min</td><Td></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>　95℃　</Td><Td>　15sec　</Td><Td>　25 cycle　</Td></Tr>
-<tr><td>55℃(GAL4) and 58℃(Except for GAL4)</td><td>30sec</td><Td>25 cycle</Td></tr><tr>
+<tr><td>　55℃(GAL4) and 58℃(Except for GAL4)　</td><td>　30sec　</td><Td>　25 cycle　</Td></tr><tr>
-<td>68℃</td><td>2min30sec</td><Td>25 cycle</Td></tr>
+<td>　68℃　</td><td>　2min30sec　</td><Td>　25 cycle　</Td></tr>
-<td>68℃</td><td>2min30sec</td><Td></Td></tr>
+<td>　68℃　</td><td>　2min30sec　</td><Td></Td></tr>
-<td>14℃</td><td>∞</td><Td></Td></tr>
+<td>　14℃　</td><td>　∞　</td><Td></Td></tr>
 </Table>
 <br><br>