Patrick

I attempted another restriction digest of cpxP in the hopes that I would get a better reading. There was, unfortunately, no separation of the bands. This suggests that all of the cpxP promoters have scarred in the plasmid, or that the plasmid had religated on itself.

Thus, I will try and do a restriction digest using EcoRI and PstI sites in orer to see if the part is released at all. If the plasmid is cut with EcoRI and PstI but not XbaI and SpeI, this definitely suggests that the part was facing in the wrong direction.

Alex

This week Thane Kubik, a former iGEM supervisor came in to provide advice in the wet lab, and help us with our project vision. Also, we did a gel of our cpxp digested product. The results of the first gel was inconclusive, the lines appeared blurry and appeared to linger in the wells, leading us to believe there was a problem with the gel itself. A second cpxp digest was done, but the spe and xba sites appeared to have not cut, as there was no separation between bands on the gel. A PCR of our cpxp plasmid was done to determine if the cpxp promoter was present in the plasmid, using the primers that we synthesized to get cpxp from native e-coli. A gel was ran of the product, and we saw feint bands at about 200 base pairs when compared to the ladder, and stronger bands at 50-200 base pairs. This was what we had anticipated as the cpxp gene we expect is 144 base pairs, and the plasmid is about 2200 base pairs.