Team:HokkaidoU Japan/Notebook/plastic Week 10

Single colony isolation

The colony of pGEM PhaCAB in JM109 was isolated to plate1 and plate2.
The colony of pGEM PhaCAB in DH5a was isolated to plate3.
Plate 1~3 was started to incubate from 18:00.

Colony PCR

We confirmed the length of constructs, [PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.
The result showed ligation to take PhaB on pSB1C3 went well.
But ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] failed.

Liquid culture

We add 2ml LB and 2ul antibiotic to ideal colony suspension and begun to cultivate at 37C, 180rpm.

Colony PCR

We confirmed the length of constructs, [RBS-PhaA on pSB1A2], [RBS-PhaC on pSB1A2] and [PhaB on pSB1C3] by colony PCR.
Ideal plasmids were liquid cultured and spreaded on LBplates.

PCR

PhaB on pSB1C3 dimer was multiplied by PCR.
And then we got DNA solution of PhaB has prefix and suffix.

Mini-prep

Mini-prep for [RBS-phaC] on pSB1A2 and [RBS-phaA] on pSB1A2 liquid culture products cultivated from 3rd. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.

Colony PCR

We confirmed the length of constructs, [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.
The result showed some of ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] went well.

Gel Extraction

Gel Extraction for digestion products. We used FastGene Gel&PCR extraction kit(NipponGenetics). Got 50 ul of DNA solution of phaB.

liquid culture

The media of pGEM phaCAB was removed to polymer producing media.

Plasmid extracton

The plasmids, [RBS-PhaC-RBS-PhaA] were extracted.
And then we got 50ul DNA solution.

Sequence

We analyzed nucleotide sequence of construct made ever.
The result showed that PhaA sequence contained a mutation.
We decided to make construct including PhaA.