Team:HokkaidoU Japan/Notebook/plastic Week 8

From 2012.igem.org

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==Colony PCR==
==Colony PCR==
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We did colony PCR of PhaA and PhaB again.
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[[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]]
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Revision as of 04:38, 24 September 2012

Contents

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

digestion result

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

Ethanol precipitation result


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...

Colony PCR result
Colony PCR result


August 24th

Colony PCR

We did colony PCR of PhaA and PhaB again.

Colony PCR result



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