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Restriction Enzyme & Gel Electrophoresis Lab

Digest DNA samples with restriction enzymes and run the fragments on a virtual agarose gel. Adjust voltage, gel concentration, and run time to see how each variable affects band migration. Build a standard curve from the DNA ladder and estimate unknown fragment sizes.

Guided Experiment: Restriction Mapping an Unknown Plasmid

You have an unknown 6 kb plasmid. If you digest it with EcoRI, BamHI, and both together, what pattern of fragments would let you determine where each enzyme cuts?

Write your hypothesis in the Lab Report panel, then click Next.

Gel Electrophoresis

Time: 0.0 / 30 minProgress: 0%
− Cathode (negative)+ Anode (positive)1kb LadderEcoRI

Controls

Voltage100 V
Gel Concentration1.0% agarose
Run Time30 min
Apply ethidium bromide stain to view band analysis and standard curve data.

Data Table

(0 rows)
#TrialBand #Distance(mm)Est. Size(bp)Actual Size(bp)% Error
0 / 500
0 / 500
0 / 500

Reference Guide

Restriction Enzymes

Restriction endonucleases are molecular scissors that cut DNA at specific palindromic recognition sequences. They are essential tools in molecular biology for cloning, mapping, and analyzing DNA.

Common enzymes in this lab
EcoRI: G↓AATTCBamHI: G↓GATCCHindIII: A↓AGCTT\text{EcoRI: G}\downarrow\text{AATTC} \quad \text{BamHI: G}\downarrow\text{GATCC} \quad \text{HindIII: A}\downarrow\text{AGCTT}

Each enzyme produces either sticky ends (5' or 3' overhangs) or blunt ends, which determines how fragments can be ligated back together.

Gel Electrophoresis

Agarose gel electrophoresis separates DNA fragments by size. DNA is negatively charged, so it migrates toward the positive electrode when voltage is applied. The agarose matrix acts as a molecular sieve.

Key variables
  • Voltage controls migration speed
  • Gel concentration affects pore size and resolution
  • Run time determines how far bands travel

Higher gel concentrations (1.5-2%) resolve small fragments better, while lower concentrations (0.5-0.8%) separate large fragments more effectively.

Standard Curve Construction

A standard curve relates migration distance to fragment size. By running a DNA ladder with known sizes alongside your sample, you can estimate the sizes of unknown fragments.

Linear relationship
log⁡10(size)=m⋅d+b\log_{10}(\text{size}) = m \cdot d + b

Plot log(bp) on the y-axis versus distance (mm) on the x-axis for each ladder band. A best-fit line through these points is your standard curve. Use it to read off the estimated size for any unknown band's distance.

Restriction Mapping

Restriction mapping uses single and double digests to determine the positions of restriction enzyme cut sites on a DNA molecule. This technique was a cornerstone of molecular biology before routine DNA sequencing.

Mapping strategy
  1. Digest with enzyme A alone and measure fragments
  2. Digest with enzyme B alone and measure fragments
  3. Digest with A + B together (double digest)
  4. Compare patterns to deduce site positions

The double digest reveals which fragments from each single digest are cut further, allowing you to construct a linear or circular map of restriction sites.