Team:Calgary/7 July 2009

• Overnight plates resulted in no colonies
• Attempted other types of ligase (Invitrogen T4 overnight at 16 degrees Celsius and NEB T4 Ligase 1 hour at room temperature) and products were transformed into Top10 cells. The cells were plated on AK/AC plates.

• Did a plasmid Isolation of Colony2X LuxOD47E BBK.

• Today I did a colony PCR with p Taq and Biobrick primers (BBK-CP-F and BBK-CP-R) to see if we were able to clone in either the J13002 promoter or the B0015 terminator to our gene of interest, BBK LuxOD47E. PCR products were run on a 1% agarose gel with LuxOD47E BBK as a size control and ddH2O as a negatve control. Gel if pictures below. Lanes 1-4 are J13002-LuxOD47E and Lanes 5-8 are LuxOD47E-B0015. Lane 9 is LuxOD47E BBK, lanes 10 and 11 are left blank and Lane 12 is a negative control with ddH2O.

• From this gel it looks like the cloning of J13002 may have been successful as the band sin the first four lanes appear slighlty higher than the band in the size control lane. We will make overnight cultures of these colonies, isolate plasmid and send the colony with the best concentration for DNA sequencing.

Purpose: Adding phosphatase to her vector. (at this point i didn't know much about her project or what she was doing. I just focused on the techniques). Next step is to ligate the insert and the vector. Finally, we transformed the plasmid into top 10 cells and plated it.

I continued to follow up with the old companies. I mostly left voicemails to the company staff who were away from the desk. I sent our sponsorship packages out to companies who expressed interest in our project.