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Team:HokkaidoU Japan/Notebook/plastic Week 12
From 2012.igem.org
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[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested with EcoRI and PstI.<br> | [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested with EcoRI and PstI.<br> | ||
[RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.<br> | [RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.<br> | ||
| - | But we failed the digestion | + | But we failed the second digestion. |
</p> | </p> | ||
| Line 104: | Line 104: | ||
The extracted DNA were condensed by EtOH precipitation. | The extracted DNA were condensed by EtOH precipitation. | ||
</p> | </p> | ||
| + | |||
| + | ==Ligation== | ||
| + | |||
| + | <p> | ||
| + | [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] were ligated too. | ||
| + | </p> | ||
| + | |||
==Liquid culture== | ==Liquid culture== | ||
<p> | <p> | ||
| - | We started to cultivate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180rpm to try digestion again. | + | We started to cultivate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180rpm to try digestion and ligation again. |
</p> | </p> | ||
| + | |||
| + | |||
| + | |||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
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<div> | <div> | ||
| + | |||
| + | |||
| + | -------------- | ||
==Plasmid extraction== | ==Plasmid extraction== | ||
<p> | <p> | ||
| - | Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] was extracted. | + | Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was extracted. |
</p> | </p> | ||
| Line 122: | Line 135: | ||
<p> | <p> | ||
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested with EcoRI and SpeI restriction sites.<br/> | [RBS-PhaC-RBS-PhaA on pSB1A2] was digested with EcoRI and SpeI restriction sites.<br/> | ||
| + | [RBS-PhaB on pSB1C3] was digested with EcoRI and SpeI.<br> | ||
| + | [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested with EcoRI and PstI.<br> | ||
| + | [RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.<br> | ||
</p> | </p> | ||
==DNA extraction== | ==DNA extraction== | ||
<p> | <p> | ||
| - | The digested DNA | + | The digested DNA were extracted. |
</p> | </p> | ||
| Line 132: | Line 148: | ||
==Ethanol precipitation== | ==Ethanol precipitation== | ||
<p> | <p> | ||
| - | The extracted DNA | + | The extracted DNA were condensed by EtOH precipitation. |
</p> | </p> | ||
| - | == | + | ==Ligation== |
<p> | <p> | ||
| - | [RBS-PhaC-RBS-PhaA | + | [RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.<br> |
| - | [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on | + | [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] were ligated too. |
| + | </p> | ||
| + | |||
| + | ==Transformation== | ||
| + | <p> | ||
| + | The ligated DNA were transformed into E.coli (strain: JM109).<br/> | ||
| + | E.Coli solution was spread on LBC. | ||
| + | </p> | ||
| + | |||
| + | ==Colony PCR== | ||
| + | <p> | ||
| + | We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] by colony PCR.<br/> | ||
| + | </p> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <div class="hokkaidou-notebook-daily"> | ||
| + | ==September 20th== | ||
| + | <div> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | ------------ | ||
| + | ==Colony PCR== | ||
| + | <p> | ||
| + | We confirmed the ligation [RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3], | ||
| + | [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] by colony PCR.<br/> | ||
| + | </p> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
| + | <div class="hokkaidou-notebook-daily"> | ||
| + | ==September 21st== | ||
| + | <div> | ||
| + | |||
| + | |||
| + | |||
| + | ---------- | ||
| + | ==Plasmid extraction== | ||
| + | <p> | ||
| + | Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted. | ||
</p> | </p> | ||
Revision as of 01:52, 25 September 2012
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 with XbaI and PstI.
pTet on pSB1A2 was also digested with 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 colony of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] was 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 with EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested with EcoRI and XbaI.
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested with EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested with EcoRI and PstI.
But we failed the second digestion.
DNA extraction
The digested DNA were extracted.
Ethanol precipitation
The extracted DNA were condensed by EtOH precipitation.
Ligation
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] were ligated too.
Liquid culture
We started to cultivate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180rpm to try digestion and ligation again.
September 19th
Plasmid extraction
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was extracted.
Digestion
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested with EcoRI and SpeI restriction sites.
[RBS-PhaB on pSB1C3] was digested with EcoRI and SpeI.
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested with EcoRI and PstI.
[RBS-PhaB on pSB1C3] was digested with 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] and [pSB1C3] by colony PCR.
September 20th
Colony PCR
We confirmed the ligation [RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3],
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and [pSB1C3] by colony PCR.
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.
