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Team:HokkaidoU Japan/Notebook/plastic Week 12
From 2012.igem.org
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| - | ====Plasmid extraction | + | ====Plasmid extraction=== |
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted. | Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted. | ||
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Revision as of 21:41, 26 September 2012
Contents |
September 17th
Plasmid extraction
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.
And then we got DNA solution of them.
Digestion
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.
pTet on pSB1A2 was also digested by SpeI and PstI.
Gel extraction
We confirmed the succession of digestion by electrophoresis.
And then DNA were extracted from TBE gel.
Ethanol precipitation
The digested DNAs were condensed by Ethanol precipitation.
Ligation
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.
Transformation
The ligated DNA was transformed into E. coli (strain: JM109).
And then we spread fungus liquid on LBA plates.
Colony PCR
We confirmed the ligation of [RBS-B] and pSB1C3. The results showed that the ligation went well.
We chose three colonies and started the liquid culture.
Liquid culture
The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.
September 18th
Colony PCR
We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.
RBS-PhaB-dT, a part of insert was multiplied.
Sequencing
The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed. The result showed that...
Plasmid extraction
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.
Digestion
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.
But we failed the second digestion.
Gel extraction
We confirmed succession of digestion by electrophoresis. And then DNA were extracted from TBE gel.
Ethanol precipitation
The extracted DNAs were condensed by Ethanol precipitation.
Ligation
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.
Liquid culture
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.
September 19th
Plasmid extraction
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.
Digestion
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.
DNA extraction
The digested DNA were extracted.
Ethanol precipitation
The extracted DNA were condensed by EtOH precipitation.
Ligation
[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.
Transformation
The ligated DNA were transformed into E. coli (strain: JM109).
E. coli solution was spread on LBC.
Colony PCR
We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.
The results showed that the ligation went well.
We chose three colonies and started the liquid culture.
September 20th
Colony PCR
We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.
The results showed that the ligation went well.
We chose three colonies and started the liquid culture.
September 21st=
=Plasmid extraction
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.
We submitted our parts and finished sending them ! Hooray!;)
September 22th
Liquid culture in large scale
We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28. Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.
| LB | mess up to 50 ml |
| 50% Glucose | 2 ml |
| Arabinose | 2.5 ml |
| Amp(100 mg/ml) | 50 ul |
| Cp | 50 ul |
Negative control
- Gulcose (-)
- Arabinose (-)
Cultured for 48 hrs.
September 23th
Transformation
Transformed BBa_R0011 for ligation of promoter with IPTG induction.
Cultured in LB+K plate.
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