cloning of an operon - (Sep/08/2010 )
Im trying to clone and express the riboflavin biosynthesis operon in E. coli. There are 4 overlapping genes and each contains it start and stop codon, and the total length of this is 8 kb. Is it possible to amplify 8 kb using Pfu?. Also is is possible to clone the operon in pET30 plasmid and express in E. coliBL21?
Hi, for your first part of question, I had used novagen KOD hotstart and cloned a 9kb genes before. If your Pfu can't make it you shall try KOD hotstart as It also have the proof reading capability.
I don't have too much experience with Pfu, but 8 kb is not that big -- I cloned a 15 kb operon using Hi-Fi Taq -- so it's possible. I don't understand why you're using a his-tag vector/strain system for this, but the PCR product is able to be cloned into any appropriately prepared vector. Whether it will be expressed in any given recipient strain of E. coli is dependent on whether it'll be toxic to the cell.