Team:HokkaidoU Japan/Notebook/plastic Week 8

Digestion

We digested 3 samples.
Digested of PhaC and pSB1C3 by XbaI and SpeI.

Digested of PhaA by XbaI site and SpeI site.

And digested PhaB by XbaI site and SpeI site.

digestion result

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.

Ethanol precipitation result

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.

Colony PCR result

Colony PCR result

Colony PCR

We confirmed whether the ligation at August 22nd went well by colony PCR result.

Colony PCR result

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.

Ligation

We ligated phaA and phaB with pSB1C3.

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.

Colony PCR result

Colony PCR result

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.

digestion result

Plasmid extraction

The plasmid of RBS (BBa_B0034) were extracted.
And then we got 50ul DNA solution.

plasmid extraction result

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.