"
Team:HokkaidoU Japan/Notebook/plastic Week 8
From 2012.igem.org
(Difference between revisions)
(→Plasmid extraction) |
|||
| (6 intermediate revisions not shown) | |||
| Line 81: | Line 81: | ||
===August 21st=== | ===August 21st=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| - | |||
[[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. | ||
| Line 88: | Line 88: | ||
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. | ||
| + | <br style="line-height: 0; clear: both;" /> | ||
</div></div> | </div></div> | ||
| Line 93: | Line 94: | ||
===August 22nd=== | ===August 22nd=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| - | |||
[[image:HokkaidoU 2012 120822PhaA PhaB pSB1C3 ETOH.jpg|thumb|Ethanol precipitation result]] | [[image:HokkaidoU 2012 120822PhaA PhaB pSB1C3 ETOH.jpg|thumb|Ethanol precipitation result]] | ||
| + | ====Ethanol precipitation==== | ||
The DNA extracted at August 21st were condensed by Ethanol precipitation. | The DNA extracted at August 21st were condensed by Ethanol precipitation. | ||
| Line 103: | Line 104: | ||
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. | ||
| + | <br style="line-height: 0; clear: both;" /> | ||
</div></div> | </div></div> | ||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
| + | |||
===August 23rd=== | ===August 23rd=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| + | [[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]] | ||
| + | [[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]] | ||
====Colony PCR==== | ====Colony PCR==== | ||
We confirmed whether the ligation at August 22nd went well by colony PCR result. | We confirmed whether the ligation at August 22nd went well by colony PCR result. | ||
| - | + | <br style="line-height: 0; clear: both;" /> | |
| - | + | ||
</div></div> | </div></div> | ||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
| + | |||
===August 24th=== | ===August 24th=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| - | |||
| - | |||
| - | |||
[[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]] | [[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]] | ||
[[image:HokkaidoU2012 120824 PhaB coloP.jpg|thumb|Colony PCR result]] | [[image:HokkaidoU2012 120824 PhaB coloP.jpg|thumb|Colony PCR result]] | ||
| + | |||
| + | ====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. | From the result, we thought that we failed to ligate PhaA with pSB1C3 and PhaB with pSB1C3. So we decided to ligated them again. | ||
| Line 128: | Line 133: | ||
====Ligation==== | ====Ligation==== | ||
We ligated phaA and phaB with pSB1C3. | We ligated phaA and phaB with pSB1C3. | ||
| + | <br style="line-height: 0; clear: both;" /> | ||
</div></div> | </div></div> | ||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
| + | |||
===August 25th=== | ===August 25th=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| + | [[image:HokkaidoU120825 phaAcolonyPCR.jpg|thumb|Colony PCR result]] | ||
| + | [[image:HokkaidoU2012 120825 phaBcolonyPCR.jpg|thumb|Colony PCR result]] | ||
| + | |||
====Colony PCR==== | ====Colony PCR==== | ||
We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.<br> | We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.<br> | ||
The result showed only PhaA and pSB1C3 were ligated correctly. | The result showed only PhaA and pSB1C3 were ligated correctly. | ||
| - | |||
| - | |||
| - | |||
====Liquid culture==== | ====Liquid culture==== | ||
| Line 147: | Line 154: | ||
[[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]] | [[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]] | ||
| + | <br style="line-height: 0; clear: both;" /> | ||
</div></div> | </div></div> | ||
<div class="hokkaidou-notebook-daily"> | <div class="hokkaidou-notebook-daily"> | ||
| + | |||
===August 26th=== | ===August 26th=== | ||
<div class="hokkaidou-section"> | <div class="hokkaidou-section"> | ||
| - | |||
| - | |||
[[image:HokkaidoU2012 120826 RBS&PhaA.jpg|thumb|plasmid extraction result]] | [[image:HokkaidoU2012 120826 RBS&PhaA.jpg|thumb|plasmid extraction result]] | ||
| + | ====Plasmid extraction==== | ||
| + | The plasmid of RBS (BBa_B0034) were extracted.<br/>And then we got 50 ul DNA solution. | ||
====Digestion==== | ====Digestion==== | ||
| Line 161: | Line 170: | ||
====Liquid culture==== | ====Liquid culture==== | ||
We started to incubate. | We started to incubate. | ||
| + | <br style="line-height: 0; clear: both;" /> | ||
</div></div> | </div></div> | ||
Latest revision as of 04:00, 27 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 50 ul 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.
