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Team:HokkaidoU Japan/Notebook/plastic Week 8
From 2012.igem.org
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==Liquid culture== | ==Liquid culture== | ||
Revision as of 05:23, 24 September 2012
August 20th
Digestion
A digestion to divide PhaC and pSB1C3 with XbaI and SpeI. </br>
| DNA solution PhaC | 12 ul |
| XbaI | 1 ul |
| SpeI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 4 ul |
| Total | 20 ul |
A digestion to divide PhaA and XbaI site and SpeI site.</ br>
| DNA solution PhaA | 7 ul |
| XbaI | 1 ul |
| SpeI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 9 ul |
| Total | 20 ul |
A digestion to divide PhaB and XbaI site and SpeI site.</ br>
| DNA solution PhaB | 7 ul |
| XbaI | 1 ul |
| SpeI | 1 ul |
| 10xM buffer | 2 ul |
| DW | 9 ul |
| Total | 20 ul |
August 21th
Electrophoresis
Electrophoresis of digested phaC and non digested phaC. To check the PCR of phaA and phaB, we also did electrophoresis of them.
Gel extraction
Gel ectraction of PhaA, PhaB, pSB1C3 digestion result. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
August 22th
Ethanol precipitation
Ligation
We ligated phaA and pSB1C3, phaB and pSB1C3.
Transformation
August 23th
Colony PCR
We did colony PCR of phaA and phaB. From the result, we can say that...
August 24th
Colony PCR
We did colony PCR of PhaA and PhaB again.
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.
August 25th
Colony PCR
The length of PhaA on pSB1C3 and PhaB on pSB1C3 were confirmed by colony PCR.
The result showed PhaA and pSB1C3 were ligated correctly.
Liquid culture
Cultivation of bacteria holds RBS (B0034) was started.
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 with XbaI and PstI.
liquid culture
