|Biomaterials Design Lab||Main project page|
Previous entry Next entry
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