How many positive clones should be checked for expression and purification?
It is recommended that several positive clones, previously identified by restriction digestion, should be analyzed for expression of the fusion protein by stained SDS-PAGE and if not detectable by Coomassie staining then western blot analysis. Mini cultures (2-10 mls) may be done to check for expression. For optimizing expression 50-100 ml cultures should be used. PCR may generate stop codons and other mutations, which may affect the expression of the fusion protein or the activity of the target protein. If a purified protein exhibits low activity, other clones should be used for purification and activity assays. The clones should eventually be sequenced. The ends of the insert can be sequenced using the vector primers provided in the kit.
