Team:KIT-Kyoto/Notebook-week4

From 2012.igem.org

(Difference between revisions)
Line 97: Line 97:
<h2>August 27th</h2>
<h2>August 27th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
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.
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.
<br>
<br>
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<h2>August 28th</h2>
<h2>August 28th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
 
-
<br><br>
 
-
<strong>*Parts</strong>
 
-
<br><br>
 
<h2>August 29th</h2>
<h2>August 29th</h2>
<br>
<br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<Table Border Cellspacing="0">
<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>
Line 205: Line 199:
<h2>August 31st</h2>
<h2>August 31st</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.
The hatched larvae were picked up and transferred to the new fly food vials and kept in the 25℃ incubator.
<br><br>
<br><br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td></Td><Td>　All samples　</Td></Tr>
<Tr><Td></Td><Td>　All samples　</Td></Tr>
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<h2>September 1st</h2>
<h2>September 1st</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
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.
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.
<br><br>
<br><br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300">
<img src="https://static.igem.org/mediawiki/2012/b/b0/0901akit.png" width="500" height="300">
<br><br>
<br><br>
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<br><br>
<br><br>
<h2>September 2nd</h2>
<h2>September 2nd</h2>
-
<br><strong>*TNFA and API2</strong>
+
<br>
-
<br><br>
+
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%.
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%.
<br><br>
<br><br>
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<h2>September 3rd</h2>
<h2>September 3rd</h2>
<BR>
<BR>
-
<strong>*TNFA and API2</strong>
 
-
<br><br>
 
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
 
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td></Td><Td>　All samples　</Td></Tr>
<Tr><Td></Td><Td>　All samples　</Td></Tr>

Revision as of 02:25, 25 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.

*Parts

　pEGFP-C2(cut)　　1uL　
　Ligation high　　2.5uL　
　dH2O　　5uL　
　Total　　7.5uL　






August 28th


August 29th


　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.

　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


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.



　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


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


　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℃　　∞　




Retrieved from "http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4"