IGEM:IMPERIAL/2006/LabCalendar/2006-8-25

Testing: T9002/J37016/J37020

 * Dr. Mann suggested culturing the cells in pure LB as opposed to LB Amp. He also suggested leaving the cultures in the 5ml tubes for the 4hr wait before pipetting into the 96-well plate to prevent contamination and to have enough air circulation for growth
 * Overnight culture has been innoculated into 16ml (in pure LB - no antibiotic) and placed in shaker at 11:30am (to be taken out at 1.30pm).
 * Measurement of OD after 2h in shaker - 1:30pm :
 * T9002: 0.732
 * J37016: 0.546
 * J37020: 0.546
 * Dilution to OD 0.1 - placed the 5mL tubes into shaker (not in the 96 well plate as before) at 2:15pm
 * Tubes taken out of shaker at 6:15pm and pipetted into 96 well plate
 * Reading of fluorescence with Wallac Victor III

Testing: S01656

 * Taken out of shaker at 1:40pm after 2 times dilution into OD 0.1
 * Spun down S01656 cultures, put into T9002 - placed 5mL tubes into shaker at 2:40pm
 * Tubes taken out of shaker at 6:40pm and pipetted into 96 well plate
 * Reading of fluorescence with Wallac Victor III

Lox PCR

 * One of the two PCR reactions continues to fail. There so far seems to be no explanation for this, and so now we will retry using PCR components from different sources, as this will tell us whether or not one of these components is a problem since they possibly could be contaminated

Electroporation

 * Electroporated J37024 (1 and 2) and J37018

Sequencing

 * Sent off J37015RS off for sequencing (used Riboswitch Primer - melting temp.=44)
 * Sent off J37020 off for sequencing (used C0062 1 Primer - melting temp.=54
 * Sent off J37023 (1) off for sequencing (used two primers that are from Dr. Mann - melting temps.=39)
 * We are sequencing J37023 (1) twice because the primers we are using are on the T-overhanging plasmid and the we want to sequence the whole of the aiiA sequence which is very large - around 750 base pairs.

Culturing

 * Cultured up J37015 for cyrogenic freezing
 * Cultured up J37015RS for cyrogenic freezing
 * Cultured up J37016 for testing and cyrogenic freezing
 * Cultured up S01656 for cyrogenic freezing
 * Cultured up T9002 for testing and cyrogenic freezing