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Lab manual: Molecular biology and Genetic Engineering (Biot 275)
Project 2-3 - Identifying Plasmid Restriction Mapping Using Agarose Gel Electrophoresis

Project 2-3 - Identifying Plasmid Restriction Mapping Using Agarose Gel Electrophoresis

Last updated

Apr 25, 2025
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- Activity 2-2 - DNA Analysis via Restriction Enzymes
- Activity 2-4 - Bacterial Transformation with Engineered Plasmid

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Learning Objectives

Explain the basic principles of agarose gel electrophoresis.
Describe how DNA size and shape affect migration through a gel.
Identify the roles of loading dye, DNA ladder, and SYBR/Gel Green.
Predict how supercoiled, linear, and nicked plasmid DNA will migrate differently.
Outline how agarose gel electrophoresis can be used to distinguish between plasmid A and plasmid B.

Definition: Term

Agarose Gel Electrophoresis: A technique that separates DNA fragments by size and shape using an electric field through a gel matrix.
Agarose: A polysaccharide derived from seaweed that forms a porous gel used to separate DNA fragments.
DNA Ladder: A molecular ruler—a standard set of DNA fragments of known sizes used to estimate the length of unknown DNA samples.
Loading Dye: A dye that contains glycerol and tracking dyes to help load DNA into wells and visualize the progress of electrophoresis.
SYBR Green / Gel Green: Fluorescent dyes that intercalate into DNA and glow under blue light, allowing visualization of DNA bands.
Supercoiled DNA: A tightly coiled plasmid form that migrates faster than linear or relaxed circular forms.
Linear DNA: DNA that has been cut with restriction enzymes, resulting in a straight, uncoiled molecule.
Relaxed Circular DNA: An open-circle form of plasmid DNA that migrates more slowly than supercoiled or linear forms.
Mutagen: A chemical agent that can cause genetic mutations (e.g., SYBR Green).
Electrophoresis Buffer (TBE): A solution that conducts electricity and maintains pH during gel electrophoresis.

Reflection Questions

Why do smaller DNA fragments move farther in agarose gel electrophoresis?
If two DNA fragments are the same length but migrate differently, what could explain the difference?
What safety precautions should you follow when handling SYBR Green?

Identifying Your P450 Plasmid Using Agarose Gel Electrophoresis

In this lab, you will identify which plasmid (A or B) contains the gene for the P450 enzyme. You’ll do this using agarose gel electrophoresis, a technique that separates DNA fragments by size and shape so we can visualize and analyze them.

Agarose Gel Electrophoresis

Agarose gel electrophoresis is a common method used in molecular biology to separate DNA fragments. The gel is made from agarose, a sugar polymer derived from seaweed, that forms a mesh-like structure when cooled. This structure creates a molecular “sieve” through which DNA molecules can move. DNA is negatively charged due to its phosphate backbone. When an electric field is applied across the gel, DNA fragments will migrate from the negative end (black) toward the positive end (red). Smaller DNA fragments can weave through the pores in the gel more easily and therefore move faster and farther than larger fragments.

DNA fragments don’t always migrate based on size alone—shape matters too. When analyzing your gel, you may see bands that represent different shapes of the same DNA molecule. Plasmid DNA can come in different forms:

Supercoiled DNA is tightly wound and very compact. It migrates the fastest through the gel.
Linear DNA, which results from cutting circular DNA with restriction enzymes, moves at a moderate speed.
Relaxed circular DNA, such as nicked plasmids, is more open and bulky, so it moves slowly.

DNA is colorless, so we need a stain to see it in the gel. SYBR Green (or Gel Green) is a fluorescent dye that slips in between DNA base pairs and glows green under blue light. This allows you to visualize the DNA bands safely without using UV light (unlike the older dye, ethidium bromide).

⚠️ SYBR Green is a mutagen, which means it can alter DNA and is potentially harmful. Always wear gloves and avoid skin contact.

To estimate the size of your DNA fragments, you’ll use a DNA ladder. This is a commercially prepared sample with known DNA fragment sizes. You can compare your unknown bands to the ladder like a ruler to determine how long each fragment is (in base pairs or kilobases).

Before loading DNA samples into the gel, you’ll mix them with a loading dye, which contains Glycerol that makes the sample sink into the well, and contains tracking dyes, like bromophenol blue, that migrate ahead of the DNA and help you monitor how far the sample has run.

Materials

0.8% agarose gel
SYBR Green (Invitrogen #S33102) or Gel Green (EmbiTec #EC-1995)
MiniOne Gel Electrophoresis System
1x TBE Buffer (Fisher Scientific #BP13334)
Digested plasmids A and B (from your previous lab)
6X Loading dye
1 kb DNA Ladder (Invitrogen #10488-085)
Micropipettes and filter tips

Procedure

Add 4 µL of 6X loading dye to your 20 µL digested plasmid sample. Mix gently by pipetting up and down.
Carefully pipette the full 24 µL of your prepared sample into the assigned well in the gel.
The instructor will load two lanes with 10 µL of 1 kb DNA ladder each. This gives you a reference to compare the sizes of your fragments.
Close the lid on the electrophoresis chamber and run the gel using the MiniOne system for approximately 20–30 minutes, or until the tracking dye has migrated halfway.
Use the MiniOne viewer to turn on the blue light and observe the glowing bands. Take a picture of your gel using your phone camera for later analysis.

⚠️ Always wear gloves when handling gels, stains, and buffers.
⚠️ Never look directly into the blue light—it can damage your eyes.
⚠️ SYBR Green and Gel Green are potential carcinogens. Avoid skin and eye contact.

Post-Lecture:

Objectives

Analyze an agarose gel to determine which plasmid contains the P450 gene.
Interpret differences in band patterns based on DNA size and structure.
Explain the role of each reagent and material used during gel electrophoresis.
Apply electrophoresis results to support conclusions about molecular identity.
Reflect on common mistakes and best practices when loading and running gels.

Discussion Questions

From your gel results, which plasmid (A or B) contains the P450 gene? How can you tell?
- Explain based on the number and size of bands. A correct digestion pattern suggests the presence of the P450 insert.
If your plasmid did not show clear bands, what could have gone wrong during the procedure?
Compare your DNA banding pattern with the ladder. What is the approximate size of your fragments?
What advantages does SYBR Green have over ethidium bromide, and what safety concerns still remain?
If the plasmids lacked a selectable marker (like antibiotic resistance), how could you identify transformed E. coli cells?
- Consider alternate strategies such as blue-white screening, fluorescent markers, or colony PCR.
Restriction Mapping Practice: You are given the following digestion results for a hypothetical plasmid X (11 kb total):
- How many times does each enzyme cut the plasmid?
- Use this information to draw a restriction map. Mark where each enzyme cuts and how far apart the sites are.

Enzyme	Fragment Sizes
EcoRI	5 kb, 6 kb
BamHI	3.5 kb, 7.5 kb
EcoRI + BamHI	1.5 kb, 2 kb, 3.5 kb, 4 kb