terrific broth mistake? - (Oct/22/2008 )
Probably I'm not sure to have used the rigth amount of K2HPO4. firts question: wath is the final concentration of K2HPO4 and
KH2PO4 in 1 L ot broth? If in my broth there is K2HPO4 0,07 M instead 0,7, which kind of problems in the expression and the integrity of my protein?
Please help me .
thank u very much
K2HPO4 = 9.4g = 53.97mM
KH2PO4 = 2.2g = 15.82mM
Total concentration of potassium phosphate buffer in terrific broth is approximately 70mM = 0.07M
Are you using 700mM or 70mM of phosphate buffer?
I am assuming that you meant 70mM as normal and by accident have used 7mM.
Reduction of phosphate buffer, cause the medium to be less buffered. As a result of acetate secretion by E.coli, the media becomes acidified rather quickly. Acidification will have an effect on the growth characteristics of the E.coli. You will get less E.coli cell mass. However in terms of protein expression, well that is a wildcard. Expression of some proteins are very dependent on the growth rate of the e coli host. One can't really say if the protein's expression will be affected.
But if there is an effect, the stressed condition within the cell caused by acidification will most likely lead to poor yield of your desired protein. Perhaps even more proteins expressed in a missfolded form.