protein expression from glycerol stock - (Jan/22/2007 )
hi
i was wondering if anyone has experienced low to no protein expression from a clone that previously produced a high level of protein. i usually use glycerol stocks of BL21 (pLysS) to grow up large cultures and induce protein for purification. however, i have a new construct (pQE30) in M15[pRep4] cells that i screened for protein expression. i picked a clone that had good expression, grew it up, yadda yadda and got tons of the protein i wanted.
subsequently, however, glycerol stocks of this clone produce almost no protein as seen on a coommassie... i tried inoculating large cultures both from glycerol stock in a starter culture with appropriate antibiotics; as well as glycerol stocks streaked on a selective plate. no can do. does this mean i'm going to have to retransform and screen for expression every time i do a protein prep? yikes.
thanks!
i was wondering if anyone has experienced low to no protein expression from a clone that previously produced a high level of protein. i usually use glycerol stocks of BL21 (pLysS) to grow up large cultures and induce protein for purification. however, i have a new construct (pQE30) in M15[pRep4] cells that i screened for protein expression. i picked a clone that had good expression, grew it up, yadda yadda and got tons of the protein i wanted.
subsequently, however, glycerol stocks of this clone produce almost no protein as seen on a coommassie... i tried inoculating large cultures both from glycerol stock in a starter culture with appropriate antibiotics; as well as glycerol stocks streaked on a selective plate. no can do. does this mean i'm going to have to retransform and screen for expression every time i do a protein prep? yikes.
thanks!
I think its always good to keep plasmid stock and retransform when you need to do expression.
i never inoculate directly from glycerol stock. plate some on the plate and inoculate fresh colony the next day.