Team:KIT-Kyoto/Notebook-week5

From 2012.igem.org

(Difference between revisions)
Line 97: Line 97:
<h2>September 4th</h2>
<h2>September 4th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.
<br><br>
<br><br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td> 9/3 GAL4 </Td><Td> 40uL </Td></Tr>
<Tr><Td> 9/3 GAL4 </Td><Td> 40uL </Td></Tr>
Line 138: Line 136:
<h2>September 5th</h2>
<h2>September 5th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
<br><br>
<br><br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr>
<Tr><Td></Td><td> EGFP </td><Td> pSB1C3 </Td></Tr>
Line 183: Line 179:
<h2>September 6th</h2>
<h2>September 6th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.
<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>
Line 230: Line 224:
<h2>September 7th</h2>
<h2>September 7th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.
The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.
<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>
Line 271: Line 263:
<h2>September 8th</h2>
<h2>September 8th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.
<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>
Line 335: Line 325:
<h2>September 9th</h2>
<h2>September 9th</h2>
<BR>
<BR>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.
<br><br>
<br><br>
-
<strong>*Parts</strong>
+
 
-
<br><br>
+
<img src="http://2012.igem.org/wiki/images/1/10/0909akit.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/1/10/0909akit.png" width="500" height="300">
<br><br>
<br><br>
Line 407: Line 395:
<h2>September 10th</h2>
<h2>September 10th</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
 
-
<br><br>
+
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.
The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.
<br><br>
<br><br>

Revision as of 02:23, 25 September 2012






September 4th


The female-virgin flies (yw) and male flies (yw) were collected for mating with the microinjected males and females.

 9/3 GAL4  40uL 
 M buffer(TOYOBO)  5uL 
 XbaⅠ(TOYOBO)  0.5uL 
 SpeⅠ(TOYOBO)  0.5uL 
 dH2O  4uL 
 Total  50uL 


 each DNA  40uL 
 3 buffer(NEB)  5uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  4.5uL 
 Total  50uL 


 9/3 GAL4  23uL 
 M buffer(TOYOBO)  10uL 
 XbaⅠ(TOYOBO)  1uL 
 SpeⅠ(TOYOBO)  1uL 
 CIP  0.5uL 
 dH2O  64.5uL 
 Total  100uL 




September 5th


The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.

 EGFP  pSB1C3 
 DNA template  40uL  23uL 
 3 buffer  5uL  5uL 
 BglⅡ  0.5uL  0.5uL 
 dH2O  4.5uL  21.5uL 
 Total  50uL  50uL 




 DNA template  40uL 
 4 buffer(NEB)  5uL 
 SpeⅠ  0.5uL 
 100×BSA  0.5uL 
 CIP  0.5uL 
 dH2O  4uL 
 Total  50uL 


 1uL 
 1uL 
 dH2O  3uL 
 Ligation high  2.5uL 
 Total  7.5uL 




September 6th


The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃ for mating with the microinjected males and females.

 All samples 
 DNA template  0.25uL 
 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  34.15uL 
 Total  50uL 


 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec 30 cycle 
  58℃  30sec 30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 




 pSB1C3(XbaⅠ and SpeⅠ cut)  1uL 
 GAL4(XbaⅠ and SpeⅠ cut)  1uL 
 dH2O  3uL 
 Ligation high  2.5uL 
 Total  7.5uL 


September 7th


The eclosed male and virgin female flies from microinjected embryos were collected and kept at 25℃ for maturation.

 All samples 
 DNA template  0.25uL 
 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  34.15uL 
 Total  50uL 


 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 








September 8th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible. The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. The female-virgin flies (yw) and male flies (yw) were collected and separately kept at 25℃.

 All samples 
 DNA template  1uL 
 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  33.4uL 
 Total  50uL 


 Temperature Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 




 All samples 
 DNA template  1uL 
 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  33.4uL 
 Total  50uL 


 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  55℃(-1) or 58℃(-2)  2min30sec  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS)  30 cycle 
  68℃  30sec(UAS) or 2min10sec(Except for UAS) 
  14℃  ∞ 


September 9th


The eclosed male and virgin female flies from microinjected embryos were again collected and kept at 25℃ for maturation. In total 83 flies were eclosed from the microinjected embryos. The calculated viability for the microinjected flies was 12.0%. The female-virgin flies (yw) and male flies (yw) were again collected and separately kept at 25℃.



 UAS  40uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  0.5uL 
 SpeⅠ(NEB  0.5uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 




 All sample 
 DNA template  1uL 
 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  33.4uL 
 Total  50uL 


 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  58℃  2min30sec  30 cycle 
  68℃  2min10sec  30 cycle 
  68℃  2min10sec 
  14℃  ∞ 




 1uL 
 1uL 
 dH2O  3uL 
 Ligation high  2.5uL 
 Total  7.5uL 


 1uL 
 dH2O  4uL 
 Ligation high  2.5uL 
 Total  7.5uL 


September 10th


The adult male and virgin female flies from microinjected embryos were mated with yw virgin female flies and yw male flies, respectively. Mated flies were transferred to the new food vials in every three days to lay eggs as much as possible.

*Parts

 HS or Act5c  40uL 
 4 buffer(NEB)  5uL 
 XbaⅠ-HF(NEB)  0.5uL 
 BamHⅠ(NEB ) 0.5uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 




 G-1 and G-2  G-3 and G-4 
 DNA template  1uL  4uL 
 10× KOD plus buffer  5uL  5uL 
 2mM dNTPs  5uL  5uL 
 25mM MgSO4  1.6uL  1.6uL 
 10P 5’ primer  1uL  1uL 
 10P 3’ primer  1uL  1uL 
 KOD plus  1uL  1uL 
 dH2O  33.4uL  31.4uL 
 Total  50uL  50uL 


 Temperature  Time  Cycle 
  95℃  2min 
  95℃  15sec  30 cycle 
  57℃(G-1 and G-3) or 59℃(G-2 and G-4)  2min30sec  30 cycle 
  68℃  2min10sec  30 cycle 
  68℃  2min10sec 
  14℃  ∞ 




 pSB1C3(PCR)  40uL 
 4 buffer(NEB)  5uL 
 EcoRⅠ-HF(NEB)  0.5uL 
 SpeⅠ(NEB)  0.5uL 
 2100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


 13uL 
 3 buffer(NEB)  2uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  2.5uL 
 Total  20uL 




 GAL4  20uL 
 4 buffer(NEB)  4uL 
 XbaⅠ(NEB)  0.7uL 
 SpeⅠ(NEB)  0.7uL 
 100×BSA  0.4uL 
 dH2O  14.6uL 
 Total  40uL 




 GAL4  40uL 
 3 buffer(NEB)  5uL 
 BglⅡ(NEB)  0.5uL 
 dH2O  4.5uL 
 Total  50uL 


 GAL4(Bgl Ⅱ cut)  40uL 
 4 buffer(NEB)  5uL 
 SpeⅠ  0.5uL 
 100×BSA  0.5uL 
 dH2O  4uL 
 Total  50uL 




 1uL 
 2uL 
 EGFP or LacZ  2uL 
 Ligation high  5uL 
 Total  10uL 


 1uL 
 2uL 
 2uL 
 Ligation high  5uL 
 Total  10uL