RECOMBINANT DNA METHODS DAY ONE

AGAROSE GEL ELECTROPHORESIS

Today, you will be pouring and running an agarose gel to purify DNA fragments for ligation. This agarose gel differs from the ones you ran during the Development module in two significant ways: 1) it is a non-denaturing gel; and 2) it is made with "low melt" agarose.

Here is the gel box before placing the gel tray/comb for pouring the agarose gel.

This photo shows an agarose gel that has been poured and allowed to solidify. When pouring the gel, the gel tray is placed perpendicular to the buffer chambers. This creates a "mold" for pouring the gel.

When running the agarose gel, the gel is turned so that it is parallel to the buffer chambers, as seen here. The blue loading dye allows you to follow the migration of your samples through the gel.

SAFETY REMINDER:
To take the photo on the left, we removed the safety lid from the gel, but you should always run your agarose gel with the lid in place!


ISOLATION OF DNA FRAGMENTS

After running your low-melt agarose gel, you will isolate the DNA fragments required for your ligation by cutting them out of the gel. These pictures illustrate the basic steps in this protocol.

After running your gel, you will bring it to one of the long-wave UV boxes (located in the Alcoves near the fume hoods). Place your gel on a piece of Saran Wrap, and have a clean razor blade ready to use.

Before turning on the UV light, protect your face and eyes with a face shield. Each "cutting station" will have a number of face shields available for your use.

When you turn on the UV light, you should be able to see the DNA fragments in the gel because of their interaction with Ethidium Bromide (EtBr) contained in the gel. The bands should be pink-orange in color.

To avoid cross-contamination of your samples, first cut the gel in thirds lengthwise (cutting down the empty lanes). Separating the gel piece containing your band of interest from the rest of the gel to makes it easier to work with.


Excise the band containing your DNA fragment of interest using the razor blade, as seen here. Removing as much excess agarose as possible and cutting the band into two or three smaller pieces will help the gel slice melt faster.


Return to Virtual Lab