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Expression of K31C Ascaris Hemoglobin in BL21 (DE3) cells. Also, Sarah was in today working on getting molar absorptivity of Ni(protoporphyrin IX). Her notebook is here
(7:45am) I centrifuged the starter cultures from the previous evening (4x25ml for 15 minutes at 4500rpm and 4C). I added 1ml each of ampicilin (100mg/ml) and hemin (40mg/ml in dimethylformamide) to each 1L of expression media. I resuspended all 4 pellets in 4 mL of fresh LB broth and redistributed the pellets to each of the 1L of expression media.
Because the OD(600) can't be accurately measured do to the presence of hemin, I have to estimate when is the right time to induce expression.
(12:00pm) I added 1mL of IPTG (0.4g in 4mL) to each expression flask to induce expression.
(3:00pm) Along with Paul, I centrifuged the expression cultures to isolate the cells (4x500mL for 15 minutes at 4500 rpm and 4C - This procedure was carried out twice). After centrifugation we resuspended the cell pellets in 50mM tris 50mM NaCl pH8. (35 mL was used to resuspend the cells from 2 of the centrifuge tubes. This was done twice). The cells were placed at -80C overnight.
This procedure was continued here