Team:HokkaidoU Japan/Notebook/plastic Week 8

From 2012.igem.org

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==August 20th==
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===August 20th===
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<div>
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<div class="hokkaidou-section">
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==Digestion==
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====Digestion====
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<p>
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We digested 3 samples.<br>
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A digestion to divide PhaC and pSB1C3 with XbaI and SpeI. </br>
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Digested of PhaC and pSB1C3 by XbaI and SpeI.
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A digestion to divide PhaA and XbaI site and SpeI site.</ br>
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Digested of PhaA by XbaI site and SpeI site.
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A digestion to divide PhaB and XbaI site and SpeI site.</ br>
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And digested PhaB by XbaI site and SpeI site.
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   |20 ul
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</p>
 
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<div class="hokkaidou-notebook-daily">
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===August 21st===
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==August 21th==
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<div class="hokkaidou-section">
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<div>
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==Electrophoresis==
+
-
 
+
-
 
+
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<p>
+
-
 
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[[image:HokkaidoU2012 120821PhaA PhaB PhaC digestion okamura.jpg|thumb|digestion result]]
[[image:HokkaidoU2012 120821PhaA PhaB PhaC digestion okamura.jpg|thumb|digestion result]]
 +
====Electrophoresis====
 +
We confirmed whether PhaC was digested correctly, and phaA and phaB were done PCR correctly by electrophoresis.
-
Electrophoresis of digested phaC and non digested phaC.
+
====Gel extraction====
-
To check the PCR of phaA and phaB, we also did electrophoresis of them.
+
We confirmed succession of digestion by electrophoresis, then DNA were extracted from TBE gel.
-
 
+
And we used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
-
 
+
<br style="line-height: 0; clear: both;" />
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+
-
</p>
+
-
 
+
-
==Gel extraction==
+
-
 
+
-
<p>
+
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Gel ectraction of PhaA, PhaB, pSB1C3 digestion result.
+
-
We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
+
-
</p>
+
-
 
+
</div></div>
</div></div>
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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===August 22nd===
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==August 22th==
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<div class="hokkaidou-section">
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<div>
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==Ethanol precipitation==
+
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<p>
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[[image:HokkaidoU 2012 120822PhaA PhaB pSB1C3 ETOH.jpg|thumb|Ethanol precipitation result]]
[[image:HokkaidoU 2012 120822PhaA PhaB pSB1C3 ETOH.jpg|thumb|Ethanol precipitation result]]
 +
====Ethanol precipitation====
 +
The DNA extracted at August 21st were condensed by Ethanol precipitation.
 +
====Ligation====
 +
We ligated phaA with pSB1C3 and phaB with pSB1C3.
-
</p>
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====Transformation====
-
 
+
The ligated DNA were transformed into E. coli (strain:DH5&alpha;).
-
==Ligation==
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E. coli solution was spread on LBC.
-
<p>
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<br style="line-height: 0; clear: both;" />
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+
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We ligated phaA and pSB1C3, phaB and pSB1C3.
+
-
 
+
-
</p>
+
-
 
+
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==Transformation==
+
-
 
+
</div></div>
</div></div>
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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==August 23th==
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===August 23rd===
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<div>
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<div class="hokkaidou-section">
-
==Colony PCR==
+
-
<p>
+
-
 
+
-
We did colony PCR of phaA and phaB.
+
-
From the result, we can say that...
+
-
 
+
[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]]
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[[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]]
-
</p>
+
====Colony PCR====
-
 
+
We confirmed whether the ligation at August 22nd went well by colony PCR result.
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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==August 24th==
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===August 24th===
-
<div>
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<div class="hokkaidou-section">
-
==Colony PCR==
+
[[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]]
-
<p>
+
[[image:HokkaidoU2012 120824 PhaB coloP.jpg|thumb|Colony PCR result]]
 +
====Colony PCR====
We did colony PCR of PhaA and PhaB again.
We did colony PCR of PhaA and PhaB again.
-
[[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]]
+
From the result, we thought that we failed to ligate PhaA with pSB1C3 and PhaB with pSB1C3. So we decided to ligated them again.
-
[[image:HokkaidoU2012 120824 PhaB coloP.jpg|thumb|Colony PCR result]]
+
-
 
+
-
From the result, we thought that we failed to ligate PhaA and pSB1C3, and PhaB and pSB1C3. So we ligated PhaA and pSB1C3, and PhaB and pSB1C3 again.
+
-
</p>
+
 +
====Ligation====
 +
We ligated phaA and phaB with pSB1C3.
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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==August 25th==
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===August 25th===
-
<div>
+
<div class="hokkaidou-section">
-
==Colony PCR==
+
-
<p>
+
-
The length of PhaA on pSB1C3 and PhaB on pSB1C3 were confirmed by colony PCR.<br/>The result showed PhaA and pSB1C3 were ligated correctly.
+
-
 
+
[[image:HokkaidoU120825 phaAcolonyPCR.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU120825 phaAcolonyPCR.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU2012 120825 phaBcolonyPCR.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU2012 120825 phaBcolonyPCR.jpg|thumb|Colony PCR result]]
-
</p>
+
====Colony PCR====
-
<div>
+
We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.<br>
-
==Liquid culture==
+
The result showed only PhaA and pSB1C3 were ligated correctly.
-
<p>
+
-
Cultivation of bacteria holds RBS (B0034) was started.
+
-
</p>
+
 +
====Liquid culture====
 +
We started to incubate bacteria holds RBS (B0034).
 +
====Digestion====
 +
We digested PhaC(BBa_K342001) by XbaI and SpeI. The result shows that the digestion was succeeded.
 +
 +
[[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]]
 +
<br style="line-height: 0; clear: both;" />
</div></div>
</div></div>
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<div class="hokkaidou-notebook-daily">
<div class="hokkaidou-notebook-daily">
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==August  26th==
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===August  26th===
-
<div>
+
<div class="hokkaidou-section">
-
==Plasmid extraction==
+
[[image:HokkaidoU2012 120826 RBS&amp;PhaA.jpg|thumb|plasmid extraction result]]
-
<p>
+
====Plasmid extraction====
-
The plasmid of RBS (BBa_B0034) were extracted.<br/>And then we got 50ul DNA solution.  
+
The plasmid of RBS (BBa_B0034) were extracted.<br/>And then we got 50 ul DNA solution.
-
</p>
+
-
 
+
-
 
+
-
==Digestion==
+
-
<p>
+
-
RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.<br/>And also PhaC (BBa_K342001) was digested with XbaI and PstI.
+
-
</p>
+
-
 
+
-
==liquid culture==
+
-
 
+
-
 
+
-
 
+
-
 
+
-
</div><div>
+
-
 
+
-
 
+
-
 
+
-
 
+
 +
====Digestion====
 +
RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.<br/>And also PhaC (BBa_K342001) was digested by XbaI and PstI.
 +
====Liquid culture====
 +
We started to incubate.
 +
<br style="line-height: 0; clear: both;" />
 +
</div></div>
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi -->

Latest revision as of 04:00, 27 September 2012

Contents

August 20th

Digestion

We digested 3 samples.
Digested of PhaC and pSB1C3 by XbaI and SpeI.

DNA solution PhaC 12 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 4 ul
Total 20 ul


Digested of PhaA by XbaI site and SpeI site.

DNA solution PhaA 7 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 9 ul
Total 20 ul


And digested PhaB by XbaI site and SpeI site.

DNA solution PhaB 7 ul
XbaI 1 ul
SpeI 1 ul
10xM buffer 2 ul
DW 9 ul
Total 20 ul

August 21st

digestion result

Electrophoresis

We confirmed whether PhaC was digested correctly, and phaA and phaB were done PCR correctly by electrophoresis.

Gel extraction

We confirmed succession of digestion by electrophoresis, then DNA were extracted from TBE gel. And we used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.

August 22nd

Ethanol precipitation result

Ethanol precipitation

The DNA extracted at August 21st were condensed by Ethanol precipitation.

Ligation

We ligated phaA with pSB1C3 and phaB with pSB1C3.

Transformation

The ligated DNA were transformed into E. coli (strain:DH5α). E. coli solution was spread on LBC.

August 23rd

Colony PCR result
Colony PCR result

Colony PCR

We confirmed whether the ligation at August 22nd went well by colony PCR result.

August 24th

Colony PCR result
Colony PCR result

Colony PCR

We did colony PCR of PhaA and PhaB again.

From the result, we thought that we failed to ligate PhaA with pSB1C3 and PhaB with pSB1C3. So we decided to ligated them again.

Ligation

We ligated phaA and phaB with pSB1C3.

August 25th

Colony PCR result
Colony PCR result

Colony PCR

We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.
The result showed only PhaA and pSB1C3 were ligated correctly.

Liquid culture

We started to incubate bacteria holds RBS (B0034).

Digestion

We digested PhaC(BBa_K342001) by XbaI and SpeI. The result shows that the digestion was succeeded.

digestion result


August 26th

plasmid extraction result

Plasmid extraction

The plasmid of RBS (BBa_B0034) were extracted.
And then we got 50 ul DNA solution.

Digestion

RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.
And also PhaC (BBa_K342001) was digested by XbaI and PstI.

Liquid culture

We started to incubate.


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