10 Chapter 10 – DNA Gel Electrophoresis

Two common restriction endonucleases  EcoRI and PstI  are provided to you in this lab procedure. To better understand how EcoRI and PstI may help you in performing your DNA fingerprinting test, first understand the “cutting” effect of a restriction endonuclease on DNA.

• Label the 5 microtubes as follows:
  • Crime Scene DNA 1 vial (CS)
  • Suspect 1 DNA 1 vial (S-1)
  • Suspect 2 DNA 1 vial (S-2)
  • Suspect 3 DNA 1 vial (S-3)
  • Suspect 4 DNA 1 vial (S-4)
  • Suspect 5 DNA 1 vial (S-5)
• Place the tubes in the rack.
• The restriction digests will take place in these tubes.
• Locate the clear microtube that contains the restriction enzyme mix and label it “ENZ” (ENZ = Enzyme mix).
• Obtain the DNA samples from your instructor. Using a fresh tip for each sample, transfer 10 µl of each DNA sample from the stock tubes into each of the corresponding labeled tubes (S-1 thru S-5).

• Close the caps on all the tubes. Mix the components by gently flicking the tubes with your finger. In the  microcentrifuge, pulse the tubes for two seconds to force the liquid into the bottom of the tube to mix and combine reactants. (Be sure the tubes are BALANCED in the microcentrifuge). If your lab is not equipped with a centrifuge, briskly shake the tube or tap the tubes on the lab bench to combine and mix the contents.
• Incubate the samples by placing the tubes in the floating rack and incubate them at 37 °C for 45 minutes. (Alternatively, the tubes can be incubated in a large volume of water heated to 37 °C and allowed to slowly reach room temperature overnight).
• After the incubation, store the DNA digests in the refrigerator until the next lab period.

Electrophoresis separates DNA fragments according to their relative size. DNA fragments are loaded into an agarose gel slab, which is placed into a chamber filled with a conductive liquid buffer solution. A direct current is passed between wire electrodes at each end the chamber. DNA fragments are negatively charged, and when placed in an electric field will be drawn toward the positive pole. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. Over a period of time smaller fragments will travel farther than larger ones. Fragments of the same size stay together and migrate in single “bands” of DNA.

• Obtain a prepoured agarose gel from your instructor, or if your teacher instructs you to do so, prepare your own gel. The gels would be 0.8% or 1.2% agarose gel.
• After preparing the gel, remove your digested samples from the refrigerator.
• Using a new tip for each sample, add 5 µl of sample loading dye “LD” to each tube.
• Close the caps on all the tubes. If a centrifuge is available, pulse spin the tubes to bring the contents to the bottom of the tube. Otherwise, tap the tubes upon a table top.
• Place the casting tray with the solidified gel in it, into the platform in the gel box. The wells should be at the (-) cathode end of the box, where the black lead is connected.
• Very carefully, remove the comb from the gel by pulling it straight up.
• Pour ~ 275 ml of electrophoresis buffer into the electrophoresis chamber, until it just covers the wells.
• Locate your lambda HindIII DNA size marker in the tube labeled “M”.
  The first sample is loaded in the well at the left hand corner of the gel. (Gels are read from left to right). A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples.
• Using a separate pipette tip for each sample, load your gel as follows:
  • Lane 1: HindIII DNA size marker, clear, 10 µl
  • Lane 2: CS, green, 20 µl
  • Lane 3: S1, blue, 20 µl
  • Lane 4: S2, orange, 20 µl
  • Lane 5: S3, violet, 20 µl
  • Lane 6: S4, red, 20 µl
  • Lane 7: S5, yellow, 20 µl

• Secure the lid on the gel box. The lid will attach to the base in only one orientation: red to red and black to black. Connect electrical leads to the power supply.
• Turn on the power supply. Set it for 100 V and electrophorese the samples for 30–40 minutes.
• Shorter lengths of DNA move faster than longer lengths.
• The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye.
• The electrical current is left on long enough to ensure that the DNA fragments move far enough across the gel to separate them, but not so long that they run off the end of the gel.
• Once the DNA has migrated far enough across the gel, the electrical current is switched off and the gel is removed from the electrophoresis tank.
• When the electrophoresis is complete, turn off the power and remove the lid from the gel box. Carefully remove the gel tray and the gel from the gel box. Nudge the gel off the gel tray and carefully slide it into your plastic staining tray (Note that the gel is very slippery).
• Pour 60 ml of Bio-Safe DNA stain into your plastic staining tray, cover with plastic wrap, and let the gel stain overnight, shaking intermittently if no rocking platform is available.
• Unaided visual examination of gels indicates only the positions of the loading dyes and not the positions of the DNA fragments. DNA fragments are visualized by staining the gel with a blue dye. The blue dye molecules have a high affinity for the DNA and strongly bind to the DNA fragments, which makes them visible. These visible bands of DNA may then be traced, photographed, sketched, or retained as a permanently dried gel for analysis.

Exercise 3 is a continuation of Exercise 2.

• Pour off the Bio-Safe DNA stain into a bottle or another appropriate container and destain the gel with 60 ml of water for ~15 minutes.
• Pour the water out of the staining tray. Follow your laboratory instruction regarding   proper disposal of the stain.
• Trim away any empty lanes of the gel with a knife or razorblade. Let the gel dry on the hydrophilic side of a piece of gel support film or in your staining tray on your lab bench for 3–5 days. When the gel is dry, continue with the Quantitative Analysis of DNA Fragment Sizes
• The laboarory activity will provide more accurate measurements. In order to make the most accurate comparison between the crime scene DNA and the suspect DNA, other than just a visual match, a quantitative measurement of the fragment sizes needs to be created. This is done below:
  • • Using the ruler, measure the migration distance of each band. Measure the distance in millimeters from the bottom of the loading well to the center of each DNA band and record your numbers in a table. The data in the table will be used to construct a standard curve and to estimate the sizes of the crime scene and suspect restriction fragments.

    

    • To make an accurate estimate of the fragment sizes for either the crime scene or the suspects, a standard curve is created using the distance (x-axis) and fragment size (y-axis) data from the Lambda/HindIII size marker. Using both linear and semi-log graph paper, plot distance versus size for bands 2–6. On each graph, use a ruler and draw a line joining the points. Extend the line all the way to the right hand edge of the graph.
    • Now check the graph that provides the straightest line that you could use to estimate the crime scene or the suspects’ fragment sizes.
    • To estimate the size of an unknown crime scene or suspect fragment, find the distance that fragment traveled. Locate that distance on the x-axis of your standard graph. From that position on the x-axis, read up to the standard line, and then follow the graph line to over to the y-axis. You might want to draw a light pencil mark from the x-axis up to the standard curve and over to the y-axis showing what you’ve done.
    • Where the graph line meets the y-axis, this is the approximate size of your unknown DNA fragment. Do this for all crime scene and suspect fragments.
    • Compare the fragment sizes of the suspects and the crime scene.