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Team:HokkaidoU Japan/Notebook/plastic Week 10
From 2012.igem.org
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We confirmed the length of constructs, [PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.<br />The result showed ligation to take PhaB on pSB1C3 went well.<br />But ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] failed. | We confirmed the length of constructs, [PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA on pSB1A2] by colony PCR.<br />The result showed ligation to take PhaB on pSB1C3 went well.<br />But ligation [RBS-PhaC on pSB1A2] with [RBS-PhaA] failed. | ||
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We confirmed the length of constructs, [RBS-PhaA on pSB1A2], [RBS-PhaC on pSB1A2] and [PhaB on pSB1C3] by colony PCR.<br />Ideal plasmids were liquid cultured and spreaded on LBplates. | We confirmed the length of constructs, [RBS-PhaA on pSB1A2], [RBS-PhaC on pSB1A2] and [PhaB on pSB1C3] by colony PCR.<br />Ideal plasmids were liquid cultured and spreaded on LBplates. | ||
| + | [[image:HokkaidoU 120904RBSphaAcolop.jpg|thumb|Colony PCR result]] | ||
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==PCR== | ==PCR== | ||
Revision as of 03:50, 24 September 2012
Contents |
September 3rd
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.
September 4th
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.
September 5th
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.
September 6th
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.
September 7th
harvesting
@Taguchi lab
The weight of new centrifuge tubes were measured.
JM109
- pantothenic acid (+) 1.5ml W:1.461 (W=tube weight with nothing inside.)
- pantothenic acid (-) 1.5ml W:1.457
- pantothenic acid (+) 0.7ml W:1.576
DH5a
- pantothenic acid (+) 1.5ml W:1.461
- pantothenic acid (-) 1.5ml W:1.461
- pantothenic acid (+) 0.7ml W:1.460
The cells were centrifuged and freeze dried for two days.
