I have two question about my cloning attempt,
1) I have cloned three different genes (originated from bacillus) in to LacZ of pSK(-) (pbluescript) vector. After transformation E.coli DH5alpha and induce with 0.5 mM IPTG there is no difference on SDS gel between Host and Clones. Cloned genes has own promotor region and Shine-dalgarno sequence. I have tried different IPTG concentration, different time of induction, different media but still I see same SDS gel profile. (Cloned vector has been checked for right orientation). What do you think about this situation. Unfortunately for my protein there is no antibody and I coudn’t make western blot.
2) As you know pSK vector has LacZ promoter region and Shine-dalgarno sequence of B-galactosidase and Cloned genes has own promotor region and Shine-dalgarno sequence. Is this situation create any problem for activity.
1) Yes I have sequenced my fragment and of course I selected my clone via white/blue screen.
2) When inducing with IPTG, Is quantity of mRNA increase?? Basically; increasing mRNA quantity cause increasing protein quantity so increasing activity of protein. I want to ask you is ribosome use SD sequence of B-gal ??
It would increase, if it has been driven by the b-glu promotor, but if the gene has its own promoter, than probably not.
BTW how large is your insert? It must be really long, if it contains also the promotor.