File:Jr pulsegen 5.jpg

Pulse generator construct K142027 was produced from device K142047 using PCR-based site-directed mutagenesis as described previously. After transformation by electroporation of purified PCR product into DH5-alpha cells, eight colonies were selected and plasmids were purified using Qiagen Miniprep kit. The eight plasmids have been co-transformed together with target plasmid K142073 into electrocompetent DH5-alpha cells and were plated on LB-agar supplemented with carbenicillin and kanamycin. Upon incubation overnight at 37C, numerous colonies appeared and one colony from each plate was streaked onto a LB-agar plate supplemented with carbenicillin and kanamycin and on a LB-agar plate supplemented with carbenicillin, kanamycin and 1mM IPTG. Cells were incubated overnight at 37C for the same duration and imaged with a Nikon D80 SLR on the UV lamp of a Kodak Gel Logic 200 imaging system.