Team:KIT-Kyoto/c6h12o6

From 2012.igem.org

(Difference between revisions)
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<h2>August 1st</h2>
<h2>August 1st</h2>
<br>
<br>
-
<strong>*TNFA and API2</strong>
+
<strong>*TNFAIP3</strong>
-
<br><br>
+
<br>
 +
<br>
 +
Composition
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
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</Table>
</Table>
<br>
<br>
 +
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<br>
<br>
-
<Table Border Cellspacing="0">
 
-
<Tr><Td>  </Td><Td> sample1 </Td><Td> sample2 </Td><Td> sample3 </Td><Td> sample4 </Td></Tr>
 
-
<Tr><Td> 10ng/μL API2-MALT1 </Td><Td> 2μL </Td><Td> 2μL </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
 
-
<Tr><Td> 10× KOD plus buffer </Td><Td> 2μL </Td><Td> 2μL </Td><Td> 2μL </Td><Td> 2μL </Td></Tr>
 
-
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td><Td> 2μL </Td><Td> 2μL </Td><Td> 2μL </Td></Tr>
 
-
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 0.8μL </Td><Td> 0.8μL </Td><Td> 0.8μL </Td><Td> 0.8μL </Td></Tr>
 
-
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td><Td> 0.6μL </Td><Td> 0.6μL </Td><Td> 0.6μL </Td></Tr>
 
-
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td><Td> 0.6μL </Td><Td> 0.6μL </Td><Td> 0.6μL </Td></Tr>
 
-
<Tr><Td> KOD plus </Td><Td> 0.4μL </Td><Td> 0.4μL </Td><Td> 0.4μL </Td><Td> 0.4μL </Td></Tr>
 
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 11.6μL </Td><Td> 11.6μL </Td><Td> 12.6μL </Td><Td> 12.6μL </Td></Tr>
 
-
<Tr><Td> total </Td><Td> 20μL </Td><Td> 20μL </Td><Td> 20μL </Td><Td> 20μL </Td></Tr>
 
-
</Table>
 
-
<br>
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
 
-
<Tr><Td> 95°C </Td><Td> 2min </Td><Td>  </Td></Tr>
 
-
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 25cycle </Td></Tr>
 
-
<Tr><Td> 50°C(sample1,3)or55°C(sample2,4) </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
 
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td> 25cycle </Td></Tr>
 
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td>  </Td></Tr>
 
-
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
 
-
</Table>
 
-
<br>
 
<h2>August 2nd</h2>
<h2>August 2nd</h2>
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<br><br>
<br><br>
 +
Composition
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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<Tr><Td>  </Td><Td> 1-2 </Td><Td> 1-3 </Td></Tr>
+
<Tr><Td>  </Td><Td> 1-2 </Td></Tr>
-
<Tr><Td> DNA sample </Td><Td> 10μL </Td><Td> 20μL </Td></Tr>
+
<Tr><Td> DNA sample </Td><Td> 10μL </Td></Tr>
-
<Tr><Td> 6×Dye </Td><Td> 2μL </Td><Td> 4μL </Td></Tr>
+
<Tr><Td> 6×Dye </Td><Td> 2μL </Td></Tr>
-
<Tr><Td> total </Td><Td> 12μL </Td><Td> 25μL </Td></Tr>
+
<Tr><Td> total </Td><Td> 12μL </Td></Tr>
</Table>
</Table>
-
<br><br>
+
 
 +
<br>
 +
<br>
<img src="http://2012.igem.org/wiki/images/e/e0/0802.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/e/e0/0802.png" width="500" height="300">
<br>
<br>
<br>
<br>
 +
 +
Composition
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
<Tr><Td>  </Td><Td> 1-2 </Td></Tr>
<Tr><Td>  </Td><Td> 1-2 </Td></Tr>
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<Tr><Td> total </Td><Td> 108μL </Td></Tr>
<Tr><Td> total </Td><Td> 108μL </Td></Tr>
</Table>
</Table>
-
<br><br>
+
 
 +
<br>
 +
<br>
 +
 
<img src="http://2012.igem.org/wiki/images/1/19/0802-2.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/1/19/0802-2.png" width="500" height="300">
<br><br>
<br><br>
<img src="http://2012.igem.org/wiki/images/0/0e/0802-3.png" width="500" height="300">
<img src="http://2012.igem.org/wiki/images/0/0e/0802-3.png" width="500" height="300">
-
<br><br>
+
<br>
-
<Table Border Cellspacing="0">
+
-
<Tr><Td> 10ng/μL API2-MALT1 </Td><Td> 5μL </Td></Tr>
+
-
<Tr><Td> 10×KOD plus buffer </Td><Td> 10μL </Td></Tr>
+
-
<Tr><Td> 2mM dNTPs </Td><Td> 10μL </Td></Tr>
+
-
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 4μL </Td></Tr>
+
-
<Tr><Td> 10P 5'primer </Td><Td> 3μL </Td></Tr>
+
-
<Tr><Td> 10P 3'primer </Td><Td> 3μL </Td></Tr>
+
-
<Tr><Td> KOD plus </Td><Td> 2μL </Td></Tr>
+
-
<Tr><Td> dH<sub>2</sub>O </Td><Td> 63μL </Td></Tr>
+
-
<Tr><Td> total </Td><Td> 100μL </Td></Tr>
+
-
</Table>
+
<br>
<br>
-
<Table Border Cellspacing="0">
+
 
-
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
+
-
<Tr><Td> 95°C </Td><Td> 2min </Td><Td>  </Td></Tr>
+
-
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 35cycle </Td></Tr>
+
-
<Tr><Td> 55°C </Td><Td> 30sec </Td><Td> 35cycle </Td></Tr>
+
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td> 35cycle </Td></Tr>
+
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td>  </Td></Tr>
+
-
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
+
-
</Table>
+
-
<br>
+
<strong>Result</strong>
<strong>Result</strong>
<br><br>
<br><br>
-
<img src="http://2012.igem.org/wiki/images/1/18/0802-4.png" width="500" height="300">
+
 
-
<br><br>
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<h2>August 3rd</h2>
<h2>August 3rd</h2>
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<br>
<br>
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<br>
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<Table Border Cellspacing="0">
+
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<Tr><Td>  </Td><Td> sample1 </Td><Td> sample2 </Td></Tr>
+
-
<Tr><Td> 10ng/μL AIP2-MALT1 </Td><Td> 2.5μL </Td><Td> 2.5μL </Td></Tr>
+
-
<Tr><Td> 10×KOD plus buffer </Td><Td> 5μL </Td><Td> 5μL </Td></Tr>
+
-
<Tr><Td> 2mM dNTPs </Td><Td> 5μL </Td><Td> 5μL </Td></Tr>
+
-
<Tr><Td> 25mM MgSO<sub>4</sub> </Td><Td> 2μL </Td><Td> 2.4μL </Td></Tr>
+
-
<Tr><Td> 10P 5'primer </Td><Td> 1.5μL </Td><Td> 1.5μL </Td></Tr>
+
-
<Tr><Td> 10P 3'primer </Td><Td> 1.5μL </Td><Td> 1.5μL </Td></Tr>
+
-
<Tr><Td> KOD plus </Td><Td> 1μL </Td><Td> 1μL </Td></Tr>
+
-
<Tr><Td> dH<sub>2</sub> </Td><Td> 31.5μL </Td><Td> 31.1μL </Td></Tr>
+
-
<Tr><Td> total </Td><Td> 50μL </Td><Td> 50μL </Td></Tr>
+
-
</Table>
+
<br>
<br>
-
 
-
<Table Border Cellspacing="0">
 
-
<Tr><Td> temperature </Td><Td> time </Td><Td> cycle </Td></Tr>
 
-
<Tr><Td> 95°C </Td><Td> 2min </Td><Td>  </Td></Tr>
 
-
<Tr><Td> 95°C </Td><Td> 15sec </Td><Td> 35cycle </Td></Tr>
 
-
<Tr><Td> 55°C </Td><Td> 30sec </Td><Td> 35cycle </Td></Tr>
 
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td> 35cycle </Td></Tr>
 
-
<Tr><Td> 68°C </Td><Td> 3min30sec </Td><Td>  </Td></Tr>
 
-
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
 
-
</Table>
 
-
<br>
 
-
<br>
 
-
<img src="http://2012.igem.org/wiki/images/d/df/0803.png" width="500" height="300">
 
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<br><br>
 
<h2>August 4th</h2>
<h2>August 4th</h2>
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<br>
<br>
<strong>*TNFA and API2</strong>
<strong>*TNFA and API2</strong>
-
<br><br>
 
-
<br><br>
 
-
<img src="http://2012.igem.org/wiki/images/2/2e/0807.png" width="500" height="300">
 
<br><br>
<br><br>
-
 
<Table Border Cellspacing="0">
<Table Border Cellspacing="0">
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</Table>
</Table>
<br>
<br>
 +
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 +
<br><br>
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<img src="http://2012.igem.org/wiki/images/2/2e/0807.png" width="500" height="300">
 +
<br><br>
 +
 +

Revision as of 04:44, 25 September 2012

August 1st


*TNFAIP3

Composition
   sample1 
 10ng/μL TNFAIP3  6μL 
 10× KOD plus buffer  10μL 
 2mM dNTPs  10μL 
 25mM MgSO4  3.2μL 
 10P 5'primer  3μL 
 10P 3'primer  3μL 
 KOD plus  2μL 
 dH2O  62.8μL 
 total  100μL 

 temperature  time  cycle 
 95°C  2min   
 95°C  15sec  25cycle 
 60°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   

August 2nd


*TNFA and API2

Composition
   1-2 
 DNA sample  10μL 
 6×Dye  2μL 
 total  12μL 




Composition
   1-2 
 DNA sample  90μL 
 6×Dye  18μL 
 total  108μL 






Result

August 3rd


*TNFA and API2

 attB TNFAIP3(80ng/μL)  1μL 
 pDONR(455ng/μL)  0.35μL 
 TE buffer  7.65 
 total  9μL 



August 4th


*TNFA and API2

August 5th


*TNFA and API2





August 6th


*TNFA and API2

August 7th


*TNFA and API2

   pDONR  BP TNFA-1  BP TNFA-2 
 DNA sample  1μL  1μL  1μL 
 6×Dye  1μL  1μL  1μL 
 dH2O  4μL  4μL  4μL 
 total  6μL  6μL  6μL 





 BP TNFA-2  2μL 
 pTFW(Destination vector)  0.5μL 
 TE buffer  6.5μL 
 total  9μL 

WEEK1終わり

August 8th


*TNFA and API2

August 9th


*TNFA and API2

 DNA sample  1μL 
 10×H buffer  0.5μL 
 EcoRⅠ  0.2μL 
 dH2O  3.3μL 
 total  5μL 




   1-2 
 50ng/μL LR TNFA-1  1μL 
 10×rTaq buffer  2μL 
 2mM dNTPs  2μL 
 25mM MgCl2  0.8μL 
 10P 5'primer  0.6μL 
 10P 3'primer  0.6μL 
 rTaq  0.4μL 
 dH2O  12.6μL 
 total  20μL 

 temperature  time  cycle 
 95°C  2min   
 95°C  15sec  25cycle 
 55°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   

August 10th


*TNFA and API2





August 13th


*TNFA and API2

 1-1  1μL 
 10×M buffer  0.5μL 
 Hind Ⅲ  0.2μL 
 dH2O  3.3μL 
 total  5μL 




August 14th


*TNFA and API2

 1-1  sample1 
 10ng/μL TNFAIP3  6μL 
 10×KOD plus buffer  10μL 
 2mM dNTPs  10μL 
 25mM MgSO4  3.2μL 
 10P 5'primer  3μL 
 10P 3'primer  3μL 
 KOD plus  2μL 
 dH2O  62.8μL 
 total  100μL 

 temperature  time  cycle 
 95°C  2min   
 95°C  15sec  25cycle 
 60°C  30sec  25cycle 
 68°C  2min30sec  25cycle 
 68°C  2min30sec   
 14°C  ∞   




WEEK2終わり

August 20th


TNFA and API2

1. Reproduction of DNA from gel
We did agarose gel electrophoresis (0.7% gel).

Composition
   a product of PCR attB TNFAIP3(8/1) 
 DNA sample  90μL 
 6×Dye  18μL 
 Total  108μL 

We separated 54uL×2 from 108uL (sample) and we did electrophoresis (54uL sample and 1kb marker 5uL).We separated gel containing DNA.

Results
We isolated DNA from these gels by QIA quick Gel Extraction Kit.
Finally we melt DNA to TE Buffer(40uL)

August 21st


TNFA and API2

1. Density measurement of attB TNFAIP3
We did agarose gel electrophoresis (0.7% gel). (1kb marker-3uL, 5times dilution attB TNFAIP3-1uL (make August 20 ), 10times dilution-1uL, 1kb marker-2uL, 15times dilution-1uL, 20times dilution-1uL, 1kb marker-1uL )

Results We estimated attB TNFAIP3(we make this time) is 35ng/uL

2. BP reaction
We adjusted solution (vials) on 1.5mL tube to next composition.
 attB TNFAIP3(35ng/μL)  2μL 
 pDONR(150ng/μL)  1μL 
 TE buffer  5μL 
 Total  8μL 

We added BP Clonase Ⅱ enzyme mix-2uL to this vials.
We incubated these vials for 2hour at 25˚C. Next we did BP reaction

3. Transformation
We added BP TNFAIP3-2uL to XL1-Blue-100uL and we did Transformation.
Finally we spread 10uL, 40uL and 100uL on plate of LB Kanamycin(+) and we keep 37℃ overnight.

August 22nd


TNFA and API2

We separated six colonies from 40uL spread plate of BP TNFAIP3 (make August 21 ) .
We cultivated these colonies on 2.5mL Kanamycin(+) LB culture medium and we kept shaking this medium overnight.