Part:BBa_K1129006:Experience

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Applications of BBa_K1129006

The Cas9 construct was transformed into E. cloni 10G, plated on LB agar plates made with chloramphenicol (33ug/mL), and grown overnight at 37C.

A single colony was picked and grown overnight in LB (with chloramphenicol) at 30C and shaking (1000 rpm). Growth was very slow for the Cas9 culture and OD was only ~0.6 in the morning. Several aliquots of 175 uL were taken and placed in separate wells of a 96-well plate. They were induced with varying concentrations of arabinose (0, 0.5%, 1%, and 2%) for three hours at 37C and 1000 rpm.

OD was measured and it was determined that there was a tradeoff between induction concentration and growth. The highest induction concentration that also had relatively good growth was 1% arabinose (OD=1.17 vs 0.98 for 2% induction and 1.67 for non-induced). This culture and the non-induced control was collected, centrifuged, resuspended in SDS-PAGE loading buffer, and boiled for 10 minutes. They were loaded in a

https://static.igem.org/mediawiki/2013/0/0a/UBC-Growth_curves_MOI.png

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