Team:KIT-Kyoto/Notebook-week2

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<h2>August 8th</h2>
 +
<br>
 +
<br>
 +
We isolated 5 colonies from the LB plate, and cultured.<br>
 +
<br>
 +
<br>
 +
 +
 +
<h2>August 9th</h2>
 +
<br>
 +
<br>
 +
 +
1. Purification of the candidate pUAS-flag-TNFAIP3 DNA<br>
 +
<br>
 +
The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.<br>
 +
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.<br>
 +
<br>
 +
<br>
 +
2. Characterization of the candidate pUAS-flag-TNFAIP3 DNA<br>
 +
<br>
 +
The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.<br>
 +
<br> 
 +
 +
<Table Border Cellspacing="0">
 +
<Tr><Td> Each diluted DNA sample </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> 10×H buffer </Td><Td> 0.5μL </Td></Tr>
 +
<Tr><Td> EcoRⅠ </Td><Td> 0.2μL </Td></Tr>
 +
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 5μL </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
 +
After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.<br>
 +
Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right.<br>
 +
<br>
 +
<br>
 +
 +
 +
<strong>Result</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/9/9b/0809.png" width="500" height="300">
 +
<br><br>
 +
The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.<br>
 +
<br>
 +
<br>
 +
3. PCR amplification of the insert DNA<br>
 +
<br>
 +
The four 5'primers were designed for PCR as follows.<br>
 +
<br>
 +
 +
・GW3r<br>
 +
5’-ATCGAGGCCTGTCTAGAGAAGC<br>
 +
・TNFA-1<br>
 +
5’-GGCTTTGCTATGATACTCGGAACTG<br>
 +
・TNFA-2<br>
 +
5’-GTAAAATGTGAAACGCCCAACTGC<br>
 +
・TNFA-3<br>
 +
5’-GGACTCCAGAAAACAAGGGCTTT<br>
 +
<br>
 +
<br>
 +
 +
 +
The 3’ primer was designed as follows.<br>
 +
・SVr<br>
 +
5’-GGCATTCCACCACTGCTCCC<br>
 +
<br>
 +
<br>
 +
 +
 +
PCR reactions with the following combinations of primers were carried out.<br>
 +
sample1→GW3r―SVr<br>
 +
sample2→TNFA-1―SVr<br>
 +
sample3→TNFA-2―SVr<br>
 +
sample4→TNFA-3―SVr<br>
 +
 +
<br>
 +
<br>
 +
 +
 +
PCR was carried out in the following reactions.
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>  </Td><Td> Each sample </Td></Tr>
 +
<Tr><Td> 50ng/μL LR TNFA-1 </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> 10×rTaq buffer </Td><Td> 2μL </Td></Tr>
 +
<Tr><Td> 2mM dNTPs </Td><Td> 2μL </Td></Tr>
 +
<Tr><Td> 25mM MgCl<sub>2</sub> </Td><Td> 0.8μL </Td></Tr>
 +
<Tr><Td> 10P 5'primer </Td><Td> 0.6μL </Td></Tr>
 +
<Tr><Td> 10P 3'primer </Td><Td> 0.6μL </Td></Tr>
 +
<Tr><Td> rTaq </Td><Td> 0.4μL </Td></Tr>
 +
<Tr><Td> dH<sub>2</sub>O </Td><Td> 12.6μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 20μL </Td></Tr>
 +
</Table>
 +
<br><br>
 +
 +
Reaction conditions of PCR
 +
<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> 55°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
 +
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
 +
</Table>
 +
<br>
 +
 +
 +
 +
 +
<h2>August 10th</h2>
 +
<br>
 +
<br>
 +
 +
Electrophoresis of PCR products was carried out.<br>
 +
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.<br>
 +
<br>
 +
Left marker5uL sample1 sample2 sample3 sample4 Wright<br>
 +
<br>
 +
 +
<strong>Result</strong><br>
 +
 +
<img src="http://2012.igem.org/wiki/images/8/84/0810.png" width="500" height="300">
 +
<br>
 +
The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.<br>
 +
<br>
 +
<br>
 +
 +
 +
 +
 +
 +
 +
 +
 +
<h2>August 13th</h2>
 +
<br>
 +
<br>
 +
 +
Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.<br>
 +
<br>
 +
The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.<br>
 +
<br>
 +
 +
 +
<Table Border Cellspacing="0">
 +
<Tr><Td> the candidate pENTR-TNFAIP3 prepared on 8/6 </Td><Td> 1μL </Td></Tr>
 +
<Tr><Td> 10×M buffer </Td><Td> 0.5μL </Td></Tr>
 +
<Tr><Td> Hind Ⅲ </Td><Td> 0.2μL </Td></Tr>
 +
<Tr><Td> dH<sub>2</sub>O </Td><Td> 3.3μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 5μL </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>After reaction, samples were electrophoresed in following order.<br>
 +
<br>
 +
Left: 1kb marker5uL cut sample <br>
 +
Right: uncut sample<br>
 +
<br>
 +
<br>
 +
<strong>Results</strong>
 +
<br>
 +
<img src="http://2012.igem.org/wiki/images/2/2a/0813.png" width="500" height="300">
 +
<br><br>
 +
Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.<br>
 +
 +
<br>
 +
<br>
 +
 +
 +
<h2>August 14th</h2>
 +
<br>
 +
<br>
 +
 +
Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.<br>
 +
<br>
 +
 +
Composition of the reaction
 +
<Table Border Cellspacing="0">
 +
<Tr><Td>  </Td><Td> sample1 </Td></Tr>
 +
<Tr><Td> 10ng/μL TNFAIP3 </Td><Td> 6μL </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> 3.2μL </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> 62.8μL </Td></Tr>
 +
<Tr><Td> total </Td><Td> 100μL </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
 +
Reaction conditions
 +
<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> 60°C </Td><Td> 30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td> 25cycle </Td></Tr>
 +
<Tr><Td> 68°C </Td><Td> 2min30sec </Td><Td>  </Td></Tr>
 +
<Tr><Td> 14°C </Td><Td> ∞ </Td><Td>  </Td></Tr>
 +
</Table>
 +
<br>
 +
<br>
 +
PCR product was applied to the 0.7% agarose gel electrophoresis.<br>
 +
<br>
 +
 +
<strong>Results</strong><br>
 +
<img src="http://2012.igem.org/wiki/images/c/c4/0814.png" width="500" height="300">
 +
<br>
 +
 +
<br>
 +
<br>
 +
 +
<div align="right"><a href="http://2012.igem.org/Team:KIT-Kyoto/Notebook-week3">>>>>>>>>>WEEK3</a></div>
 +
</div>
 +
</div>
 +
</table>
 +
</td></tr>
 +
 +
</table>
 +
</body>
 +
 +
 +
 +
</html>

Latest revision as of 04:13, 26 September 2012






August 8th



We isolated 5 colonies from the LB plate, and cultured.


August 9th



1. Purification of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNAs from five independent sample -1,-2,-3,-4 and -5 were purified by the alkaline-lysis method.
We added 0.2uL of 10ng/uL RNase A to them and incubate them for 1 hour.


2. Characterization of the candidate pUAS-flag-TNFAIP3 DNA

The candidate pUAS-flag-TNFAIP3 DNA was digested wit EcoRⅠ as follows.

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


After incubation of these samples for 1hour, we added 1uL of 6×Dye to each of them and electrophoresed in 0.7% agarose gel in following order.
Left, 10fold dilution, 2uL marker, 20fold dilution, 30fold dilution, 1uL marker, 40fold dilution, 0.5uL marker right.


Result


The concerntration of the candidate pUAS-flag-TNFAIP3 DNA was estimated as 200ng/uL.


3. PCR amplification of the insert DNA

The four 5'primers were designed for PCR as follows.

・GW3r
5’-ATCGAGGCCTGTCTAGAGAAGC
・TNFA-1
5’-GGCTTTGCTATGATACTCGGAACTG
・TNFA-2
5’-GTAAAATGTGAAACGCCCAACTGC
・TNFA-3
5’-GGACTCCAGAAAACAAGGGCTTT


The 3’ primer was designed as follows.
・SVr
5’-GGCATTCCACCACTGCTCCC


PCR reactions with the following combinations of primers were carried out.
sample1→GW3r―SVr
sample2→TNFA-1―SVr
sample3→TNFA-2―SVr
sample4→TNFA-3―SVr


PCR was carried out in the following reactions.
   Each sample 
 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 


Reaction conditions of PCR
 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



Electrophoresis of PCR products was carried out.
After adding 2uL of 6×Dye to each 10uL of sample, we electrophoresed them in 0.7% agarose gel in following order.

Left marker5uL sample1 sample2 sample3 sample4 Wright

Result

The sizes of the PCR products were unexpected ones. We therefore concluded the construction of pUAS-flag-TNFAIP3 DNA was not successful.


August 13th



Cut check of the plasmid DNA(the candidate pENTR-TNFAIP3) after BP reaction.

The candidate pENTR-TNFAIP3 was digested with HindⅢ that will cut out the insert DNA.

 the candidate pENTR-TNFAIP3 prepared on 8/6  1μL 
 10×M buffer  0.5μL 
 Hind Ⅲ  0.2μL 
 dH2O  3.3μL 
 total  5μL 


After reaction, samples were electrophoresed in following order.

Left: 1kb marker5uL cut sample
Right: uncut sample


Results


Insert size was different from the expected one. We therefore concluded that the construction of entry DNA was not successful.


August 14th



Based on the results, we decided to carry out the PCR reaction for TNFAIP3 DNA again.

Composition of the reaction
   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 


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


PCR product was applied to the 0.7% agarose gel electrophoresis.

Results