Pulsed Field Gel Electrophoresis

Question 1

What is the advantage of using PFGE over a regular gel?

Answer 1

Can resolve larger fragments and intact chromosomes

Question 2

How do you prepare a sample to use in PFGE?

Answer 2

Centrifuge cell culture, then remove the supernatant and wash the cells in the pellet. Add liquid agarose into the cell culture and pipet it into a mold that forms gel plugs the size of a well on the running gel. Let the plugs soak into proteinase K which will break open the cell walls and digest the membranes, but leave the chromosomes intact. Then put the plug into the well of the gel

Question 3

How do you run a PFGE gel?

Answer 3

The wires are arranged in an octagon around the gel box, and a power box operates them automatically. Each wire is pulsed for milliseconds and the chromosomes migrate through the gel. Each pulse lets the DNA reorient itself, so we get better resolution

Question 4

Why would we want to use PFGE?

Answer 4

We can probe the gel for a gene and we can figure out which chromosome its on