Team:KIT-Kyoto/Notebook-week3p

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                 <li><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p"><img src="http://2012.igem.org/wiki/images/3/31/Side_week4kit.jpg" width="150" height="30"></a></li>
                 <li><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week4p"><img src="http://2012.igem.org/wiki/images/3/31/Side_week4kit.jpg" width="150" height="30"></a></li>
                 <li><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week5p"><img src="http://2012.igem.org/wiki/images/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li>
                 <li><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week5p"><img src="http://2012.igem.org/wiki/images/e/e7/Side_week5kit.jpg" width="150" height="30"></a></li>
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                <li><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week6p"><img src="http://2012.igem.org/wiki/images/e/e8/Side_week6kit.jpg" width="150" height="30"></a></li>
 
             </ul>
             </ul>
         </li>
         </li>

Revision as of 10:21, 25 September 2012






September 6th


 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


 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


 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




 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


 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 


September 11th


 pSB1C3-UAS-1, -2, -3, -4  5uL 
 3buffer(NEB)  2uL 
 BglⅡ  0.5uL 
 dH2O  12.5uL 
 Total  20uL 




 GAL4  40uL 
 4 buffer(NEB)  5uL 
 XbaⅠ(NEB)  0.5uL 
 SpeⅠ(NEB)  0.5uL 
 CIP  0.5uL 
 100×BSA  0.5uL 
 dH2O  3uL 
 Total  40uL 




 1uL 
 2.5uL 
 2.5uL 
 Ligation high  6uL 
 Total  12uL 


 2uL 
 2.5uL 
 1.5uL 
 Ligation high  6uL 
 Total  12uL 


September 12th


Composition
 pSB1C3-UAS-1, -2  40uL 
 3buffer(NEB)  5uL 
 SpeⅠ  1uL 
 100×BSA  0.5uL 
 dH2O  3.5uL 
 Total  50uL 


The digested DNA were applied to agarose gel electrophoresis. We isolated DNA from the gel by using QIA quick Gel Extraction Kit.

Photo of Agarose gel.


1-1-48 and 2-2-6 Ligation
DNA fragment carrying EGFP or LacZ was ligated into the BglII and SpeI digested pSB1C3-UAS DNA .for 1hour at 16℃.

Ligation reactions are listed below.
 pSB1C3-UAS (double digested with BglII and SpeⅠ)  1uL 
 EGFP fragments or LacZ fragments  2uL 
 EGFP or LacZ  2uL 
 Ligation high  2.5uL 
 dH2O  2uL 
 Total  7.5uL 


We ligated DNA fragment carrying GAL4 and DNA fragments carrying HS promoter or Act5c promoter-enhancer to pSB1C3 DNA for 1hour at 16℃.

Composition
 pSB1C3 (PCR) (double digested with XbaⅠand SpeI)  2uL 
 GAL4(double digested with BglⅡ and SpeⅠ)  2uL 
 HS fragments or Act5c fragments (double digested with XbaⅠand BamHⅠ)  2uL 
 Ligation high  6uL 
 Total  12uL 


1-1-49 and 2-2-7 Transformation of E. coli by Ligation products
We did transformation of E. coli Xl-1 blue with each of ligation products. Finally, we spread them on the LB Chloramphenicol(+) plate and incubated for 16 hours at 37℃.