How Does the NEB PCR Cloning Kit Work?

What are toxic mini-genes, and how do they improve transformation efficiencies? Becky explains.

Script

Becky:
It's really a very simple phenomenon because basically what we did is we took this circular plasmid that has this mini-gene and then we cut it right in the middle of that two amino acid mini-gene.

The problem with Klenow cloning isn't getting fragments in. It's keeping the vector that doesn't have an insert from closing up without an insert, and that's what has to be reduced.

But in this way, with a toxic mini-gene that's been cut right in the middle where the researcher puts their insert, if it does this and closes up, it's a lethal event for the cell. And so you never get a colony on a plate.

So what we have developed for our customers is a method where they can clone into this interrupted mini-gene and the only events that will lead to cell growth, which leads to a colony on a plate, are going to be the ones that have an insert interrupting it, because this is fatal, this with an insert in the middle is not. So it just makes it easier that when they get their plate and they look at it and they see colonies, what they see is what they want.

Loading Spinner