A lot of colonies have grown with the reaction with only BBa B0015 and in the reaction with BBa B0015 + BBa K112806, we selected 15 to do a Colony PCR, none of them was positive. These results show that coudn't do a good digestion of BBa B0015, so tha's why it's recircularizing and giving a lot of fake positives.
To improve our digestion we let BBa B0015 digesting ON with EcoRI and tomorrow we will purify it from agarose gel to make sure we only get linearized plasmid, then we will digest with XbaI tomorrow.
Marcos
YeastGuard
We confirmed the insertion of pJEN1 in pGEM by colony PCR using the part's internal forward primer and the vector's reverse primer (M13). Unfortunately there were no transformed colonies with JENorf, neither lysozyme.
GEL
We continued screening JENorf and Lysozyme! To find positive colonies we screened 10 more colonies of each plate and made a backup inoculum in case we find right ones. We found 7 positive colonies for lysozyme, unfortunately no positive colonies were found for JENorf again.
GEL
GEL
We did colony PCR to amplify the fragment between the vector's annealing sites (vector's primers forward and reverse) that we expected to be the YFP linked to the terminator sequence. We found some strange fragments in YFP-End PCR. This fragaments correspond to the vectors size (YFP Biobrick) without the insert (End). We believe that this fragment correspond to the recircularized vector. Our expected fragment wouldn't be amplified, since it has 3000pb, so we decided to perform miniprep from the colonies that showed no amplicons! We hope we are right! =)
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