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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==Digestion==
==Digestion==
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We digested three samples.<br>
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We digested 3 samples.<br>
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Digestions of PhaC and pSB1C3 with XbaI and SpeI. <br>
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Digested of PhaC and pSB1C3 by XbaI and SpeI. <br>
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Digestion of PhaA with XbaI site and SpeI site.<br>
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Digested of PhaA by XbaI site and SpeI site.<br>
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And digestion of PhaB with XbaI site and SpeI site.<br>
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And digested PhaB by XbaI site and SpeI site.<br>
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==August 21st==
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==August 21st==
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==August 22nd==
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==August 22nd==
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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]]
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The DNA extracted yesterday were condensed by Ethanol precipitation.
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The DNA extracted at August 21st were condensed by Ethanol precipitation.
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The ligated DNA were transformed into E.coli (strain: _____).
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The ligated DNA were transformed into E. coli (strain:DH5&alpha;).
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E.Coli solution was spread on LB_.
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E. coli solution was spread on LBC.
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==August 23rd==
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==August 23rd==
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==Colony PCR==
==Colony PCR==
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<p>
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We confirmed whether the ligation of yesterday went well by colony PCR.
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We confirmed whether the ligation at August 22nd went well by colony PCR result.
[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]]
[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]]
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==August 24th==
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==August 24th==
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==Colony PCR==
==Colony PCR==
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==August 25th==
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==August 25th==
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==Colony PCR==
==Colony PCR==
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==Digestion==
==Digestion==
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We digested PhaC(BBa_K342001) with XbaI and SpeI. The result shows that the digestion succeeded.
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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]]
[[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]]
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==Digestion==
==Digestion==
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RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.<br/>And also PhaC (BBa_K342001) was digested with XbaI and PstI.
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RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.<br/>And also PhaC (BBa_K342001) was digested by XbaI and PstI.
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Revision as of 15:54, 26 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

Electrophoresis

digestion result

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

Ethanol precipitation result

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

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

Colony PCR result
Colony PCR result


August 24th

Colony PCR

We did colony PCR of PhaA and PhaB again.

Colony PCR result
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.


Ligation

We ligated phaA and phaB with pSB1C3.


August 25th

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.

Colony PCR result
Colony PCR result

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

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

plasmid extraction result


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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