I want to clone a gene into a vector and then western blot for the production of my protein after inducing expression. Do I need to include a RBS site sequence in my forward primer or will a primer with the restriction sites I will be using and the coding domain sequence (starting with the start codon) work fine?
If your clonig vector plasmid has a RBS in the right distance to the start codon of your orf of interest it will work. Without a RBS you will not get translation of the protein. Many vector plasmid have RBS but sometimes you have the to clone in frame adding few amino acids.
You speak about inducing, so i suppose that you are working with an inducible promotor (Gal1?). If that's the case I suppose that your plasmid already as this region and so you just have to be sure that you clone your ORF of interest in frame with that promotor. If you are thinking on cloning the ORF under its endogenous promotor, than you have to choose a cloning vector that does not have already promotor region and clone the upstream region as well. Hope it helps
So i have cloned into the pUT18 vector which is used for bacterial two hybrid experiments. Upstream of the MCS there is lacZ which contains an RBS. i have cloned my gene into this plasmid and looking at the protein sequence I see the start codon of the lacZ and then there is a stop codon and then a few amino acids and then the start codon of my protein of interest which i have cloned. Even though before this start codon there was a stop codon will my protein still be translated?
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