Dear all,
I have just purchased a BL21 auxothrophic strain (RF12) from Addgene. The cells come in the form of agar stab. I intend to grow more cells which will be ready to used for protein expression. I am wondering if I should grow the cells in LB media at 37 deg until OD~0.37, then treat the cells with CaF2 in a way like treating standard BL21 competent cells?
I appreciate your guidence ! Thank you !
DEar Xiu
if i well understand those BL21 RF12 strain is auxotrophic for tryptophan, therefore in theory it can growh well in complex media as LB, that contains free aminoacids while will not growth in chemically defined media with out the supplementation of the specific aminoacid.
Therefore i think that it can grow well in the LB up the low OD that you need for competent cells preparation.
However, if you are afraid that the amount of thryptophan in the LB is low, you can certanilly improve its growt ability by supplement the LB with 50-100mg/l of tryptophan.
good luck
Manuele
I agree with Manuele Martinelli , there should be no reason why you shouldn't use LB and a standard protocol for making competent cells.
As you need to first re-streak your cells on an agar plate, you can immediately test LB +/- Trp and see if there is any growth difference. In case there isn't, this can save you some material (Trp) when doing larger cultures for competent cells.
Good luck!
BL21 auxothrophic strain cell can have many fold increase ln viability when chemically complemented with the ligand benzothiazole
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