Science Buddies: "Ask an Expert"

I have the DNA sequence for porcine pepsin and I want to find the restriction enzymes that can make the sticky ends. Once I find the correct enzyme without cutting the DNA sequence I can put it into pGLO, but the enzymes need to be the same for both. I don't know if there is a website?

Hi BrandyLC44,

I think you are asking how to pick enzymes that will not cut your DNA sequence but will work in the pGLO plasmid system right? First, you need to have the multiple cloning sight of the plasmid to know which restriction enzymes you can use. Here is a link to the pGLO plasmid map that contains the multiple cloning sight http://www.snapgene.com/resources/plasm ... mids/pGLO/. Next, you need to make sure you dont pick restriction sights that cut your DNA insert. I like to use programs such as EnzymeX: http://nucleobytes.com/index.php/enzymex or the NEBcutter: http://tools.neb.com/NEBcutter2/ Try one of those to pick a restriction enzyme that will not cut your DNA insert and is in the multiple cloning sight of the pGLO vector. When you find an enzyme that works than you will need to clone the restriction sites onto the 5' and 3' ends of your DNA insert.

Let me know if you have anymore questions! Goodluck

What we mean is if we can buy a DNA sequence with a gene,in the middle, that already has restriction sites at the ends?

Hum...I haven't heard of that option. We generally just do it ourselves. Let me know if you need any help designing primers etc.