Cloning in BioBrick vectors
From 2007.igem.org
We have amplified with PCR our promoters and Pho80 coding sequence and we have loaded 1µl on the gel to see if there were aspecific products or others. 1ORE-CYCtata
In the same time we have transformed plasmid pBS1A3[1] in competent cells and after MIDI-prep we have loaded 1µl on the gel. We have digested in two differents ways our plasmid to be sure of it's identity! Then we have digested it with XbaI and SacII and we have loaded all digest product on gel to separate by gel extraction the insert from plasmid!
We decided to clone Pho80 coding sequence in pBS1A3 plasmid but since this cds have two XbaI restriction sites in 93 and 648 positions we mutated this sites and after we cloned this gene in pBS1A3 plasmid using XbaI and SpeI enzyme for digestion of Pho80 mutated and pBS1A3 plasmid. than we used EcoRI and SpeI enzyme to digest pBS1A3-Pho80 mutated and we cloned this gene in pBca1020-r0040 upstream to rfp.
