Team:HokkaidoU Japan/Notebook/plastic Week 8
From 2012.igem.org
August 20th
Digestion
We digested three samples.
Digestions of PhaC and pSB1C3 with XbaI and SpeI.
DNA solution PhaC | 12 ul |
XbaI | 1 ul |
SpeI | 1 ul |
10xM buffer | 2 ul |
DW | 4 ul |
Total | 20 ul |
Digestion of PhaA with 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 digestion of PhaB with 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
August 24th
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 cultivate bacteria holds RBS (B0034).
Digestion
We digested PhaC(BBa_K342001) with XbaI and SpeI. The result shows that the digestion succeeded.
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
We started to cultivated.