Running a Cellulose Acetate Allozyme Gel


The plants are growing in "cone-tainers".  Select four different plants to sample.

Place a small piece of leaf in each of four separate wells of a spot plate.  Add 200 ul of grinding buffer.  Keep on ice!  Two groups will share a spot plate.

Using a different grinding tube for each depression, grind the leaf until it is completely liquified.

Remove 10 ul of solution from a well.  Try to avoid getting non-liquid parts of the leaves.

Pipet the liquid into one of the wells of the loading tray.  Each plant must go into a separate well.  Two groups will fill up one loading tray.

The "super Z" applicator is a delicate and expensive instrument and must be handled gently.  The sample is suspended between the paired wires for each lane.

The gel consists of a thin layer of cellulose acetate stuck to the surface of a plastic sheet.  It will be soaked in buffer before use.  You can tell which side the gel is on because its surface is dull.

The back side is shiny.  Do not put this side up! 

Gently blot the gel between paper towels to remove the excess buffer.  Do not rub the gel - the cellulose acetate will easily rub off. 

The gel should be placed on the loading guide so that it is lined up with the "cathode application" line. 

The super Z applicator fits into the slots of the loading guide. Make sure that the numbers on the applicator match those on the loading guide.

The applicator also fits in the slots of the loading guide.

 

Two gels can be put on the gel rig.  Be sure to orient the origin end of the gel toward the black (negative) end of the rig.  Place a paper wick on each side of the gel so that one end lays flat across the end of the gel and the other hangs down into the buffer tank. 

Place the lid on the rig so that the black plug fits over the black prong. 


The gel will be stained by the lab staff using substrate and stain suspended in agar.  Caution: the stain mixture is carcinogenic.  Do not handle the gel with your bare hands!

After about ten minutes, the development of the stain will begin to reveal the location of the enzymes.  (The agar has been removed in this image to make the bands more apparent.  On your gel, you will leave the agar in place when you read it.)

Reading a gel

By convention, the origin of allozyme gels is at the bottom.  The genotypes of the plants in each gel are listed below the place where the sample was stamped.