Harvard BioDesign/11 June 2014

From 2014.igem.org

Preparing electrocompetent cells

Picked two 5mL LSR10 inoculated cultures and added them each to a 500mL Low Na LB broth flask. Flasks were left shaking at 37C and periodically cell density was measured using nanodrop. Once cell density reached ~0.8 for each flask (we wanted ~0.7), they were placed on ice in the cold room and procedure for preparing electrocompetent cells followed. The procedure was as follows: we spun down the cells, removed the supernatant, and then washed with 10% glycerol. This was repeated three times, using less and less glycerol each time. Once we had the cells resuspended in about 30 ml of glycerol, we transferred them to 1 ml aliquots and put them in the -80C. This entire process was done on ice (spins were at 4C).

Congo red spin down assay

Put 1 ml of culture in an eppendorf. Spun that down, resuspended in PBS. Then we added .015% Congo red solution and spun again. We then looked at our cells on the plate reader at 490 nm: