Team:WashU/Week10

Monday, July 30

Failed PCR in morning

Digestion of Z construct and tet-resistant plasmid with E and S. Run on gel

PCR using KlenTaq - worked [PICTURE]

Transformed GC5 cells with Z-construct and U Plated cells to check tomorrow

Tuesday, July 21

Colony PCR on TOPO Ligation of Z construct to T plasmid PCR of Z and U constructs

Wednesday, August 1
Double transformation of GC5 cells with zeaxanthin-producing construct and TOPO PCR #2, done with a control vector as well Today we conducted a colony PCR of the TOPO cloning of ZCD with the fusion protein (without a his-tag). Colony PCR showed a single band of approximately 1200bp for the sample of colony number two. A successful colony PCR should yield amplification of ZCD with a 60bp upstream region, for a total of 1170bp. The close agreement between the band size and the expected band suggest that colony number 2 had the correct orientation of the insert (gel below). This colony will be grown up overnight and miniprepped in the morning.

Thursday, August 2
Miniprep of colony 2 from yesterday

Double transformation of GC5 cells with the miniprepped colony and zeaxanthin producing construct. Will induce with proper amounts of arabinose and IPTG at optimal concentrations once OD600 reaches 0.4

Friday, August 3