Cloning a specific gene in a plasmid vector

Question

Please explian in details ( or refer to a paper ,website etc) the steps required to clone a specific gene e.g (Colicin gene, a mobilizable plasmid like RSF1010) into a conjugative plasmid .

The Questions that I dont know about gene cloning in a plasmid are:

1. How do we make a restricition site into gene to be cloned? and do we alwayes need to do that ?

2. Does it matter if the gene cloned downstream or upstream of any other gene ; e.g ( ori gene or antibiotic marker gene ... Etc..) or Should We clone in it in a specific site in order to be properly translated?

3. How do we detect the presence of the cloned gene after we do the cloning , I prefer to put another antibiotic marker but i dont know how exactly to do it

Thanks alot

Answer 1

1. Regularly, you don't need make RS in gene. So, you need design couple of specific PCR primers for the gene ends. Primers can carry specific sequences of RSes for cloning outside of parts are complementary to gene.

2. Yes, you should. Downstream promoter.

3. By PCR with specific primers. If you need to have product would be better to get sequence of insertion. By antibiotic markers you can determine presence or absence of any insertion in target plasmid on screening agar dishes. It is primary screening. Second step is cutting of inserted gene by specific site, if it have any, or PCR and digestion of PCR product by specific site(s). This variant is preferable because solve all questions about results.