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Team:HokkaidoU Japan/Notebook/plastic Week 11
From 2012.igem.org
September 10th
PCR
PhaA was multiplied from pGEM with XbaI and SpeI restriction sites by PCR.
Digestion
PhaA was digested with XbaI and SpeI. RBS on pSB1A2 was digested with SpeI.
Gel extraction
We confirmed succession of digestion by electrophoresis.
And then DNA were extracted from TBE gel.
Ligation
RBS on pSB1A2 was ligated with PhaA and PhaB.
Transformation
These ligated DNAs transformed into E.coli (strain: DH5α).
And then we spread fungus liquid added LB on plates.
September 11th
sequence
We analyzed the sequence which contains phaA to see if there is a mutation.
Colony PCR
The colony of RBS+phaB on 1A2 was
September 12th
Digestion
RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested with EcoRI and XbaI.
ligation
The fragment of RBS-PhaB and dT(B0015) on pSB1AK3 was ligated.
However, because of my stupid mistake, both fragment failed to ligate.
I deeply feel sorry to Mr.Kawata and the E.coli.
polymer extraction
@Taguchi lab
The dried cells were removed to glass test tubes.
Weight of plastic tubes were measured in order to estimate how much the cells weighed.
Chloroform was added to the dried cells and was incubated on 60C for 48hrs.
September 13th
Digestion
We were worried about yesterdays lab work, so we tried it again.
RBS-PhaB on pSB1A2 was digested with EcoRI and SpeI restriction sites and dT(B0015) on pSB1AK3 was also digested with EcoRI and XbaI.
Gel extraction
We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.
Ethanol precipitation
The DNA solution of RBS-PhaB and dT on pSB1AK3 were concentrated by EtOH precipitation.
Ligation
RBS-PhaB was ligated with dT on pSB1AK3.
We added LB to fungus liquid mixed ligated DNAs and then spread it on LBK plates.
September 14th
Digestion
To submit our construct as a BioBrick, we had to ligate our part samples in pSB1C3.
RBS-phaC on pSB1A2, RBS-phaA and pSB1C3 was digested by EcoRI and PstI.
Gel extraction
We confirmed the succession of digestion by electrophoresis, and extracted the DNAs from TBE gel.
polymer extraction
The samples were filiterized and was removed to another test tubes. I made a 1.7mL loss of sample2.
Colony PCR
We confirmed whether or not RBS-PhaB was ligated with dT on pSB1AK3 correctly by colony PCR.
Liquid culture
We added 2ml LB with 2ul kanamycin to pre-cultured colony suspension at 37C.
And then started to cultivate at 37C, 180rpm.
