Team:WashU/Week10

Monday, July 30

We did a PCR, but failed in the morning.

Meanwhile, we executed digestion of Z construct and tet-resistant plasmid with E and S. Afterward, we ran the digested plasmid on gel electrophoresis.

Then, we did a PCR on the digest plasmid using KlenTaq and we obtained the gel using UV-Vis. Please see the picture below.

Furthermore, we have transformed GC5 cells with Z-construct and U Plated cells. We will check those tomorrow.

Tuesday, July 21

Today, we did Colony PCR on TOPO and ligation of Z construct to T plasmid. Meanwhile, we did PCR of Z and U constructs.

Wednesday, August 1
We did a double transformation of GC5 cells with zeaxanthin-producing construct and TOPO PCR #2, and we also did that transformation with a control vector. 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 suggests 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. Meanwhile, we also did a colony PCR of the U-construct. We checked the ligation of U construct and colonies of Z construct.

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 Separated out culture to have one at 20 degrees and one at 37, will induce both of them and see what happens 20 degrees limits inclusion bodies

Also ran a colony PCR on three different U colonies with two controls

Friday, August 3