Exercise 20: Gel Electrophoresis

Introduction

Gel electrophoresis is a technique used by scientists to separate genetic material based upon its size. Deoxyribonucleic acid (DNA) is an acid and therefore a negatively charged molecule. If an electrical current is applied across a buffered chamber the DNA will be drawn toward the positive terminal (see Figure 20.1). Electrophoresis uses this property to separate DNA fragments as they move through a 1.5% agarose gel. The agarose polymerizes forming a network of fibers through which the DNA moves. The gel also contains a special dye (ethidium bromide) that stains the DNA fragments making them visible under UV light. When an electrical field is applied the smaller pieces of DNA will migrate faster towards the positive end of the gel, and bigger pieces will be left behind. Different DNA fragments in a sample can “race” against each other creating a banding pattern that is like a “fingerprint” for that sample. The banding patterns of multiple samples can be compared to determine the relatedness between samples. The presence or absence of specific sized bands can identify the presence of a specific DNA target – such as a gene or plasmid. Gels always include at least one lane that includes a DNA ladder containing specific sized fragments. Sample bands can be compared to the ladder bands to estimate the size of the DNA fragment.

In this exercise students will load samples of their PCR product from exercise 19 on a gel along with a DNA ladder. If the target DNA sequence was present in the sample, PCR should have amplified the target creating millions of copies (DNA fragments). The presence of a DNA band in the sample lane indicates that the sample was positive for the target DNA. If there is no band in that sample, amplification did not occur indicating that sample was negative for the target DNA (see Figure 20.2).

Figure 20.2 Long Description

Course Intended Outcomes

Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Micropipettes
• Sterile tips
• Agarose gel inside mold
• Gel box with running buffer and power module
• Additional materials and cultures will be assigned by your instructor

Procedure

Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Wear gloves and protective eye wear at all times!
3. Do NOT touch the gel, buffer, pipettes, or tubes of DNA with bare hands
4. Carefully remove the gel mold caps and comb from the gel.
5. Place the gel (including the support piece) into the electrophoresis chamber (Figure 20.2). Position carefully so that the tab on support slides into the notch in the chamber making sure that it is level.
6. Allow your DNA sample to thaw for a few minutes. Keep on ice until the gel is loaded.
7. Use the preset 3.0 µL micropipette to add 3 µL loading dye to a small piece of paraffin paper that has been taped to the counter.
8. Use the preset 7.0 µL micropipette to add 7 µL PCR sample DNA on top of the drop of loading dye. Be careful to draw the exact amount of sample as they are shared with ALL the groups.
9. Mix slowly by gently pipetting in and out a few times.
10. Switch to the 10.0 µL pipette to accommodate the mixture volume and slowly draw up the mix holding the pipette at a slight angle.
11. Pipette all of your mixture into one of the wells in the gel.
12. Immerse your tip in the buffer, but do NOT poke the tip into the gel, the mixture should sink down into the well
13. Repeat steps 6 – 11 for lanes 2 through 5.
14. In lane 6 load 7 µL of DNA ladder
15. Place the lid on the gel box making sure that the positive terminal (red) is at the bottom of the gel. Then turn on the power and press the <run> button on the power module for the gel.
16. When the loading dye has separated and moved three-quarters of the way down the gel the run can be stopped. Turn off the power unit. Remove the gel (wear gloves!) and place on the UV light box. Close the lid and turn UV light on to observe the bands.

Sampling/Inoculation:

1. Your instructor will assign the samples to be used by your group.
2. Collect the required materials for the lab.
3. Draw an illustration of your gel in your lab notebook. Carefully label lanes 1 – 6 with the samples used for future reference.

Lab Clean-up

All pipet tips should be disposed into the sharps box.

Gels should be thrown away in the biohazard container when everyone in the group has had a chance to view the results.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Complete the results table below.

Critical Thinking Questions

If a plasmid had an additional gene inserted, what effect would you expect it to have on its representative band on a gel?

If your expected PCR product should be 50KB, but on your gel you notice a band a 20KB would you consider your sample/specimen positive or negative? Explain your answer.