cloning past stop codon - 3'end stop codon cloning (Nov/18/2008 )
I am about to clone a yeast gene into a pRS314 plasmid. I know approximately how much upstream sequence I want to include, but have no clue about downstream of the stop codon. What happens if I only clone the promoter, ORF, and stop codon? Will I get proper gene termination? If not, how much sequence 3' of the stop codon should I use?
There is no strict rule as to how much upstream and downstream sequence to use for cloning. I usually try to add at least 150 bp on each end if possible. You should look at the distance from adjacent genes to guide how much to use. Id use as much as possible.