Electrophoresis

Laboratory Activity 2.3

Introduction

The third phase of the activity is the identification of the DNA sequence through electrophoresis. This technique will help
you identify the types of DNA present. This is intended for confirmation of samples. Please walk through the experience
through the virtual lab provided to you. You are to supply the next steps of the process by following the outline.

MATERIALS:
 gel mold
 masking tape
 ethanol
 agarose
 weigh boat
 250 ml Erlenmeyer flask
 1X Tris/Borate/EDTA buffer
 magnetic stir bar
 stir plate
 Pipette
 gel rig
 light box

PROCEDURES:

 Wipe the gel mold with ethanol. Then, seal the ends of the mold with just regular masking tape
 Wrap it up just like a Christmas present.
 Push the ends down to make sure that it doesn’t leak when we pour the agarose. And do the same thing to the other
end.
 Put the comb in immediately.
 Once you put the comb you move the whole thing this out of the way for a minute to make the regular agarose gel.
 Put 0.32 grams of regular agarose in a little weigh boat and pour it into the Erlenmeyer flask.
 Also, add 1X Tris/Borate/EDTA buffer into the Erlenmeyer flask. At the same time wash out the weigh boat with
the buffer to get the remaining agarose that are stuck into it.
 to melt the agarose, take the Erlenmeyer flask into the microwave and microwave it on high about forty seconds
until it boils. Then, pour the gel and let it cool down.
 Put magnetic stir bar that's been sterilized into the flask and then turn the stir plate to its lowest setting. (it takes 2
or 3 minutes for that to cool down)
 While it’s cooling down, get the pipette then put in 64 microliters of the loading dye into the flask.
 After the dye got evenly dispersed. Take the flask out of the stir plate and get a couple large magnetic stir bars to
anchor the small one that's inside and slowly pour the agarose gel into the mold
 (That'll take twenty or thirty minutes to set)
 So, the 0.8% regular agarose gel took twenty or thirty minutes to set and harden.
 Put it down into the gel rig that has the same buffer that you make the gel with the 1X TBE buffer.
 (It's been sitting in there for a couple of minutes because we want the buffer to be able to get around the teeth of
the comb, so that we can easily extract the comb,)
 Put in the DNA that has been digested overnight using restriction enzymes and then going to run it.
 Now it’s time to take the comb out make sure that your anchoring, holding down the mold
 Put 10 microliters of ladder into the well of the mold down into the second stop.
 Get a fresh tip for every sample. Slowly, load the enzyme PST1 into the second wall
 The third vial was digested with PST1 and a second restriction endonuclease.
  Put the leads on black to black and red to red (do the same in front to connect the leads)
 Run the gel between 100 and 120 volts and press the run button (this can run between 45 and 60 mins.)
 When you see the small bond of bromophenol blue that would be the near of the end of the gel and that's when you
going to turn it off.
 It’s time to look your bands on the light box. Slide it right off onto the light box so that you can look at the bands
so, that you can see the DNA ladder.
 Now is the time to visualize the DNA

FINISHED PRODUCT/S:
 DNA ladder- the standard of known sizes

APPLICATION OF THE PRODUCTS:

 for DNA fingerprinting to investigate crime scenes. To analyze results of polymerase chain reaction. To analyze
genes associated with a particular illness. In DNA profiling for taxonomy studies to distinguish different species.

ADVERSE REACTIONS IF ERRORS ARE COMMITTED DURING THE PROCESS:

 faint or no bands on the gel/ Too little DNA can be hard to detect on a gel.