Cloning in BioBrick vectors

From 2007.igem.org

(Difference between revisions)
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We have amplified with [[PCR]] promoters and Pho80 coding sequence and we have loaded 1µl on the agarose gel to see if there were amplification products,aspecific products or others.Then we have digest our PCR products with XbaI and SpeI.
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We have amplified with [[PCR]] promoters and Pho80 coding sequence and we have loaded 1µl on the agarose gel to see if there were amplification products,aspecific products or others.Then we digested our PCR products with XbaI and SpeI.
[[1ORE-CYCtata]]
[[1ORE-CYCtata]]
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We have digested in two differents ways our plasmid to be sure of it's identity!
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We digested in two differents ways our plasmid to be sure of its identity!
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Then we have digested [[pSB1A3]][http://2007.igem.org/Biobrick_Vector_choice] with the same enzyme used to inserts.
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Then we digested [[pSB1A3]][http://2007.igem.org/Biobrick_Vector_choice] with the same enzyme used for the insert.
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We have choiced plasmid that containing one insert of 1,5kb so towards gel extraction we have separated the linearized plasmid from insert.
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We chose a plasmid containing one insert of 1,5kb so via gel extraction, we were able to separate the linearized plasmid from the insert.
[[Image:pBs1A3.jpg]]
[[Image:pBs1A3.jpg]]
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After we have realized the ligations between pSB1A3 and 1ore,2ore and fox3.
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We performed ligations between pSB1A3 and 1ore,2ore and fox3.
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After ligations we have transformed plasmid pSB1A3 in competent cells and after mini&maxi-inoculo and MIDI-prep we have loaded 1µl on the agarose gel.
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After ligations, we transformed plasmid pSB1A3 in competent cells and after mini&maxi-inoculations and MIDI-prep we loaded 1µl on the agarose gel.
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[[PHO80cds]]
[[PHO80cds]]
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We decided to clone Pho80 coding sequence in pSB1A3 plasmid but since this cds have two XbaI restriction sites in  
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We decided to clone Pho80 coding sequence in pSB1A3 plasmid but since its CDS has two XbaI restriction sites in positions
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91 and 648 positions we mutated this sites.After we cloned this gene in pSB1A3 plasmid using XbaI and SpeI enzymes for [[digestion]] of Pho80 mutated and pSB1A3 plasmid.
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91 and 648 we mutated these sites. We then cloned this gene in pSB1A3 plasmid using XbaI and SpeI enzymes for [[digestion]] of Pho80 mutated and pSB1A3 plasmid.
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After this ligations we used EcoRI and SpeI enzymes to digest pSB1A3-Pho80 casset and to clone its upstream to Rfp in pBca1020-r0040 vector.
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After this ligation, we used EcoRI and SpeI enzymes to digest pSB1A3-Pho80 cassette and to clone it upstream of Rfp in pBca1020-r0040 vector.

Revision as of 16:47, 25 October 2007

We have amplified with PCR promoters and Pho80 coding sequence and we have loaded 1µl on the agarose gel to see if there were amplification products,aspecific products or others.Then we digested our PCR products with XbaI and SpeI.

1ORE-CYCtata

2XORE-CYCtata

FOX3promoter


We digested in two differents ways our plasmid to be sure of its identity! Then we digested pSB1A3[1] with the same enzyme used for the insert. We chose a plasmid containing one insert of 1,5kb so via gel extraction, we were able to separate the linearized plasmid from the insert.

PBs1A3.jpg

We performed ligations between pSB1A3 and 1ore,2ore and fox3. After ligations, we transformed plasmid pSB1A3 in competent cells and after mini&maxi-inoculations and MIDI-prep we loaded 1µl on the agarose gel.


PHO80cds

We decided to clone Pho80 coding sequence in pSB1A3 plasmid but since its CDS has two XbaI restriction sites in positions 91 and 648 we mutated these sites. We then cloned this gene in pSB1A3 plasmid using XbaI and SpeI enzymes for digestion of Pho80 mutated and pSB1A3 plasmid. After this ligation, we used EcoRI and SpeI enzymes to digest pSB1A3-Pho80 cassette and to clone it upstream of Rfp in pBca1020-r0040 vector.