Cloning in BioBrick vectors
From 2007.igem.org
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.
In the same time we have digested pBS1A3[1] with the same enzyme used to inserts.
We have choiced plasmid that containing one insert of 1,5kb so towards gel extraction we have separated the linearized plasmid from insert.
After we have realized the ligations betwwen pBS1A3 and 1ore,2ore and fox3. After ligations we have transformed plasmid pBS1A3 in competent cells and after mini&maxi-inoculo and MIDI-prep we have loaded 1µl on the agarose gel. We have digested in two differents ways our plasmid to be sure of it's identity! Then we have digested pBS1A3 with XbaI and SpeI and we have loaded all digest product on agarose gel to separate by gel extraction the insert ccdB from plasmid!
We decided to clone Pho80 coding sequence in pBS1A3 plasmid but since this cds have two XbaI restriction sites in 91 and 648 positions we mutated this sites.After we cloned this gene in pBS1A3 plasmid using XbaI and SpeI enzymes for digestion of Pho80 mutated and pBS1A3 plasmid. After this ligations we used EcoRI and SpeI enzymes to digest pBS1A3-Pho80 casset and to clone its upstream to Rfp in pBca1020-r0040 vector.
