Re: When to use Directional or Non-directional subcloning?

When subcloning a gene of interest into a plasmid for gene expression, the 
directionality of cloning is of extreme importance.  In order for your 
gene to transcribed into mRNA correctly, your gene must be situated with 
its 5' end following a promoter (use of the correct promoter is very 
important and depends on which organism you are trying to express your 
protein).  Remember, DNA is is transcribed into mRNA in the 5' to 3' 
direction, so your start (ATG) codon must follow the promoter.  In some 
instances, there are promoters in plasmids flanking the multiple cloning 
sites and RNA transcript can be made in either direction, depending on 
which RNA polymerase is used (this is mostly used for in vitro 
transcription/translation reactions).