IGEM:IMPERIAL/2006/LabCalendar/2006-9-4

 TO DO: *Testing of T9002, J37016, J37020, S01656, J37015, J37015RS *Culture J37036 & J37018 from plate in incubator for maxi & mini on Tuesday *Make frozen stock of J37022 & culture up for testing ! *Send J37024 for sequencing !!! *Order new maxiprep kit !!! *Need more autoclaved flasks !!! *Need more isopropanol too!

Ligations

 * J04500 (vector) and J37024 (insert)being ligated overnight

LoxP PCR

 * Was done for the smaller fragment
 * Success to be determined tomorrow

Western Blot

 * 3 colonies of Rosetta and 3 colonies of BL21 containing S01656 were cultured up.
 * The samples were recultured for a couple of hours to bring them back to an exponential growth phase
 * The samples were pipetted into 2 sets of Eppendorfs, one of which was induced with IPTG
 * The Western blot was run, and the gel was left to stain
 * The results will be known tomorrow morning

Maxiprep

 * Maxiprep new AiiA gene from overnight culture.
 * Not very much bacteria (hence DNA) present.

AiiA PCR

 * Using the new primers and the newly arrived AiiA gene (maxi-preped this morning), PCR carried oput to add the LVA tag and immunotag.
 * PCR annealing temperatures calculated to be 10 cyles at 51 and 20 cycles at 69

Culturing

 * S01656 +IPTG, S01656 -IPTG, J37016 was cultured for testing
 * J37036 & J37018 & T9002 could not be cultured since no colonies could be seen on the plate - they have to grow overnight

Frozen Cells

 * Made a frozen stock of cells: J37025 (3 different colonies, but all of them have been verified to be correct)