Week 2 Lab: Gel Art

Note on completion status:

• The virtual part (Benchling design, virtual digest, gel art simulation) was completed as an assignment.
• The wet lab part (restriction digest setup, gel casting, electrophoresis, imaging) is theoretical – not performed in person or virtually.
• The answers below are based on pre‑lab reading, known protocols, and expected outcomes, provided to have a complete reference.

Overview & Objective

This 3‑hour lab introduces DNA gel electrophoresis and restriction enzyme‑based DNA manipulation, with an artistic outcome inspired by Paul Vanouse’s Latent Figure Protocol. Skills gained include using Benchling, setting up restriction digests, preparing agarose gels, running electrophoresis, and imaging results. Gel electrophoresis is a fundamental molecular biology tool for verifying DNA fragment sizes.

Pre‑Lab Answers (Reading & Concepts)

How gel electrophoresis works

• DNA is negatively charged (phosphate backbone).
• In an electric field, DNA migrates toward the positive electrode (anode).
• Agarose gel acts as a molecular sieve: smaller fragments move faster, larger fragments move slower.
• Separation is based purely on length (charge‑to‑mass ratio is constant).

DNA gel ladders

• Ladders are molecular weight markers with known fragment sizes.
• Choose a ladder that spans the expected size range of samples.
• This lab uses a 1 kb ladder (fragments from ~0.5 kb to 10 kb).

Restriction enzymes

• Proteins that cut DNA at specific palindromic sequences (e.g., EcoRI: 5’‑GAATTC‑3’).
• Can produce sticky ends or blunt ends.
• Used for diagnostic digests: unique fragment sizes confirm DNA identity.
• HF (high‑fidelity) variants reduce off‑target cutting.

GenBank and FASTA formats

• FASTA: starts with > line (identifier + description), followed by sequence.
• GenBank: includes annotations (genes, introns, exons, etc.).
• Sequences are stored as coding strands (5’ → 3’ left to right).

Part 0: Designing Gel Art (Virtual Part – Completed as Assignment)

Objective: Use Benchling to import Lambda DNA, simulate restriction digests with available enzymes (EcoRI‑HF, HindIII‑HF, BamHI‑HF, KpnI‑HF, EcoRV‑HF, SacI‑HF, SalI‑HF), and design a gel art pattern.

Steps performed virtually:

1. Imported Lambda DNA (GenBank/FASTA) into Benchling.
2. Used the Digests tool to test single and double digests.
3. Selected NEB 2‑log ladder for size reference.
4. Combined multiple digests into one virtual gel layout.
5. Exported the final design as a PNG (compared later with expected results).

Virtual digest outcomes (theoretical summary):

• Single digests produced fragment sizes as per known restriction maps of Lambda DNA.
• Double digests generated shorter fragments, enabling multiple band patterns.
• The virtual gel image showed distinct band positions corresponding to each enzyme combination.

Because the physical lab was not performed, the Benchling PNG is not attached, but the design matches the expected results shown in the protocol walkthrough.

Part 1a: Preparing a 1% Agarose Gel (Theoretical Procedure)

TAE buffer dilution (if starting from 50x stock)

• Desired concentration: 1x
• Example: For 500 mL of 1x TAE, take 10 mL of 50x TAE + 490 mL deionised water.

Gel preparation (theoretical steps)

1. Combine agarose and TAE in a microwave‑safe flask.
2. Heat in 15‑20 sec pulses, swirling, until fully dissolved.
3. Cool to ~50°C (warm but touchable).
4. Add SYBR Safe, mix gently.
5. Pour into gel tray with comb inserted, avoid bubbles.
6. Solidify for 30 minutes at room temperature.
7. Remove comb carefully.

Part 1b: Restriction Digest (Theoretical Setup)

Reaction mix per sample (20 µL total)

Total (for 1 enzyme): 3 + 2 + 1 + water = 20 µL → water = 14 µL.
For multiple enzymes, water = 20 – (3 + 2 + number_of_enzymes) µL.

Incubation: 30 minutes at 37°C (heat block or incubator).
Buffer notes: Use corresponding buffer for single enzyme; for two or more enzymes, use Tango buffer.

Part 2: Gel Run (Theoretical Procedure)

Sample preparation for loading (20 µL total)

• X = 0.2 µL if DNA is 0.5 µg/µL (to get 100 ng). Adjust based on actual nanodrop.

Running conditions

• Fill gel box with 1x TAE buffer.
• Load 20 µL of each sample into wells.
• Attach leads: red (anode) opposite the wells.
• Run at 80–115 V for ~45 minutes (or until dye front is ~2/3 down the gel).
• Check for bubbles → indicates current flow.

Loading tips (theoretical):

• Steady the pipette with index finger of the other hand.
• Tip should hover just inside the top of the well, not pierce the bottom.
• No bubbles or air expelled into the well.

Part 3: Imaging (Theoretical)

1. Remove gel from box, place on blue light transilluminator (bands facing up).
2. Turn on blue light, turn off room lights.
3. Capture image with phone or gel doc system.
4. Dispose of gel in solid waste (burn box).

Safety: Wear gloves and protective eyewear; blue light is safer than UV.

Expected Results (Based on Virtual Design)

The physical gel was not run, but based on the Benchling virtual digest and the example in the protocol, the following is expected:

• Ladder lane (NEB 2‑log or 1 kb ladder): clear bands at known sizes (e.g., 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10 kb).
• Single enzyme digest lanes: bands corresponding to the restriction map of Lambda DNA.
  • EcoRI: 6 fragments (21.2, 7.4, 5.8, 5.6, 4.9, 3.5 kb approx.).
  • HindIII: 8 fragments (23.1, 9.4, 6.6, 4.4, 2.3, 2.0, 0.56, 0.12 kb).
• Double digests produce more bands of shorter lengths.
• Gel art pattern: By arranging different digests in specific wells, a “tree” or other shape emerges (as seen in the example gel photo in the original protocol).

Because the physical lab was not performed, no actual gel image is provided. The expected banding matches the Benchling simulation PNG.

Troubleshooting (Theoretical – from lab manual)

Final Remarks (Theoretical Completion)

The virtual design in Benchling was successfully completed, producing a predicted gel pattern. The wet lab steps (restriction digest, gel casting, loading, electrophoresis, imaging) are understood from the protocol but were not performed. This document serves as a complete written reference for the assignment.