Week 2 Lab: DNA Gel Art

Design (Benchling)

Designed a face: eyes in lanes 1/3/7, eyebrows in lanes 2/6, nose in lane 5, lips in lane 4.

Benchling face design Digest table screenshot

Digest setup (per lane, 20 µL total)

Lane	Enzyme(s)	Water	CutSmart 10×	λ DNA (0.5 µg/µL)	Enzyme(s) (1 µL each)
1	PvuII + SalI	13 µL	2 µL	3 µL	1 + 1 µL
2	BamHI + XhoI	13 µL	2 µL	3 µL	1 + 1 µL
3	PvuII + SalI	13 µL	2 µL	3 µL	1 + 1 µL
4	SalI only	14 µL	2 µL	3 µL	1 µL
5	NdeI + PvuII	13 µL	2 µL	3 µL	1 + 1 µL
6	BamHI + XhoI	13 µL	2 µL	3 µL	1 + 1 µL
7	PvuII + SalI	13 µL	2 µL	3 µL	1 + 1 µL

Incubated at 37 °C, 30 min.

Predicted gel (Benchling virtual digest, NEB 2-Log ladder)

Predicted gel pattern

Bench setup

Enzymes, λ DNA, 6× loading dye, and rCutSmart buffer kept on ice. Visible on ice: λ DNA, SacI, KpnI (×2), BamHI, FD PvuII (FastDigest), rCutSmart, Eco32I (= EcoRV), 6× LD.

Enzymes on ice

Gel prep & run

1% agarose: 0.75 g agarose in 75 mL 1× TAE → microwave in 15 s pulses → cool to ~50 °C → 7.5 µL SYBR Safe (10,000×) → pour with 12-well comb → set 30 min.
Load: 20 µL digest + 3.33 µL 6× loading dye per well.
Run: 80–115 V, ~45 min in 1× TAE on EC-103.

Loading the gel Partner loading Setup ready to run

Actual result

Gel did not match the predicted pattern. Bands were either absent, smeared, or in unexpected positions. The face design was not recoverable.

Actual gel

Failure analysis — most likely causes

Buffer × enzyme mismatch (top suspect). Ice bucket shows FD PvuII (Thermo FastDigest) but digests used rCutSmart (NEB). FastDigest enzymes are optimized for Thermo’s FastDigest buffer; activity in CutSmart is partial. PvuII appears in 4 of 7 lanes (1, 3, 5, 7) — one mismatch collapses most of the design.
NdeI / XhoI not confirmed on the bench. Neither tube visible on ice. If a substitute was grabbed, lanes 2, 5, 6 wouldn’t cut as designed.
Run voltage / time. Smearing and blended rows = classic too-fast / too-long signature. 115 V is on the high end; try 70–90 V.
DNA overload. 1.5 µg per digest × 20 µL loaded ≈ 150 ng/well, over the 100 ng/well guideline.
Incomplete digestion. 30 min is the floor; 60 min helps with non-optimal buffers.

Next time — checklist

Cross-check enzyme list on the bench before finalizing the Benchling design.
If using FastDigest enzymes, use FastDigest buffer, not CutSmart. Don’t mix systems.
NEB Double Digest Finder for any 2-enzyme combo.
Nanodrop the λ DNA; dilute so each lane loads ≤ 100 ng.
Run at 80–90 V for 45–60 min; stop when dye front is ~⅔ down.
Photograph the gel with the ladder lane clearly visible.