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Team:HokkaidoU Japan/Notebook/plastic Week 8
From 2012.igem.org
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===August 21st=== | ===August 21st=== | ||
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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. | We confirmed whether PhaC was digested correctly, and phaA and phaB were done PCR correctly by electrophoresis. | ||
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We confirmed succession of digestion by electrophoresis, then DNA were extracted from TBE gel. | 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. | And we used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. | ||
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</div></div> | </div></div> | ||
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The ligated DNA were transformed into E. coli (strain:DH5α). | The ligated DNA were transformed into E. coli (strain:DH5α). | ||
E. coli solution was spread on LBC. | E. coli solution was spread on LBC. | ||
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</div></div> | </div></div> | ||
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[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]] | [[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]] | ||
[[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]] | [[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]] | ||
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====Ligation==== | ====Ligation==== | ||
We ligated phaA and phaB with pSB1C3. | We ligated phaA and phaB with pSB1C3. | ||
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[[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]] | [[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]] | ||
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</div></div> | </div></div> | ||
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====Liquid culture==== | ====Liquid culture==== | ||
We started to incubate. | We started to incubate. | ||
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</div></div> | </div></div> | ||
Revision as of 20:07, 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
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
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.
August 24th
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
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.
August 26th
Plasmid extraction
The plasmid of RBS (BBa_B0034) were extracted.
And then we got 50ul 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.
