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="http://2012.igem.org/wiki/images/b/b0/0901akit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/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℃  ∞