Team:Calgary/22 July 2009

• Prepared sterilized LB broth for growing cells. Followed procedure found in the protocol page for making competent cells.
• After completion of making competent cells, transformed ligated products (pCS26+promoters ligated using NEB Quick Ligase) into XL Gold Ultracompetent cells. These cells were plated on LB+Kan agar plates and was incubated overnight at 37^oC.
• As well, as a control, we transformed pBluescript into both XL Gold Ultracompetent cells and Top 10 Cells and plated cells on LB plates to incubate overnight at 37^oC.

• Did a miniprep of overnight cultures and took concentrations.
• Set up a Restriction Digest for verification (that the B0015 terminator is present in my contruct) with XbaI and PstI. If successful, we expected to see bands at about 1.7 kb and 3 kb for the contruct (J13002-LuxOD47E-B0015) and the psb1ac3 vector respectively.

Lane 1- J13002-LuxOD47E-B0015 C5, cut with XbaI and PstI
Lane 2- C5 uncut
Lane 3- C6 cut
Lane 4- C6 uncut
Lane 5- C7 cut
Lane 6- C7 uncut
Lane 7- C8 cut
Lane 8- C8 uncut
Lane 9- Size control- J13002-LuxOD47E
Lane 10- Size control- BBK LuxOD47E

• Analysis: From this gel it looks like colonies 5, 6 and 7 may have worked (contain the B0015 terminator) as we see two bands at the expected sizes. Colony 8 has some random bands, so I will ignore this one. Colony 6 looks the most promising as it is the cleanest (no random bands). I will proceed by sending this colony down for sequencing.

• Wrote out a lab blog for this week that I shall post tomorrow morning.