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Gel Loading & Ladder Helper

Recommend the optimal DNA ladder, compute loading-dye volumes, estimate gel run time, and generate a well layout for 8/16/24/48-well mini and midi gels.

Mouse Colony ManagementGenotypingClient-Side

Try it out

Load example gel loading helper data to see the full workflow

Gel Setup

Volume of PCR product per well.

Result

Recommended Ladder
100 bp DNA Ladder
Range: 100–1,500 bp
Gels required
1
15 sample wells per gel (1 ladder lane)
Per-well load
6.0 µL
5 µL sample + 1.0 µL 6× dye
Estimated run time
88 min
5 V/cm, 1.5% agarose
Agarose note: 1.5% agarose is standard for genotyping bands in the 500 bp–1 kb range.

Well Layout (First Gel)

L (100 bp DNA Ladder)
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
—
—
—
Ladder Sample Empty

Bench Protocol

Gel loading plan for 12 samples on 1 × 16-well mini gel (1.5% agarose).

Per-sample loading mix:
  • 5.0 µL PCR product
  • 1.0 µL 6× loading dye
  • Total per well: 6.0 µL (well capacity: 20 µL)

Ladder: 100 bp DNA Ladder (100–1,500 bp), 5 µL per lane, 1 lane per gel.

Run conditions:
  • 5 V/cm × 7 cm = 35 V
  • Estimated run time: 88 min (until dye front reaches ~70% of gel length)
  • Check at 44 min for dye front position
  • Planning a genotyping gel for a batch of PCR products
  • Choosing the right ladder for an unfamiliar band size
  • Determining how many gels you need for a large batch
  • Training new techs on loading volumes and run conditions
  • Checking that your sample + dye volume fits in your gel wells

Don't use for

  • For PAGE (polyacrylamide) gels — different loading volumes and run conditions
  • For pulse-field gel electrophoresis (PFGE) — entirely different apparatus
  • For capillary electrophoresis or fragment analysis — no gel wells involved

Agarose gel electrophoresis for genotyping

How it works

DNA is negatively charged at neutral pH. When you apply an electric field, DNA fragments migrate through the agarose matrix toward the positive electrode (anode). Shorter fragments move faster because they encounter less frictional resistance. The agarose concentration sets the pore size — higher concentrations create smaller pores that slow everything down but improve resolution of small fragments.

The loading dye does two things

1. It adds density (glycerol or Ficoll) so the sample sinks into the well instead of floating away. 2. It includes tracking dyes (bromophenol blue, xylene cyanol, or Orange G) that migrate at known rates so you can see when to stop the gel. Bromophenol blue runs at roughly the same speed as a 300 bp fragment in 1% agarose; xylene cyanol runs with ~4 kb fragments.

Why the ladder matters

A DNA ladder is a set of known-size fragments that you run alongside your samples. By comparing your band's position to the ladder, you estimate its size. Choose a ladder whose densest band spacing covers your expected band size — a 100 bp ladder has bands every 100 bp from 100-1500, while a 1 kb ladder has bands at 250, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 8000, and 10000 bp.

Common gel electrophoresis problems

Smearing instead of bands

Usually means degraded DNA (old proteinase K in the lysis step, or too many freeze-thaw cycles) or overloaded wells. Reduce sample volume or re-lyse with fresh proteinase K. Can also be caused by loading too much loading dye — the glycerol smears if it is >2× the standard 1× concentration.

Bands curve across the gel ("smile" effect)

Caused by uneven heating — the center of the gel gets hotter and the DNA migrates faster there. Lower the voltage or move the gel to a cold room. For wide midi-gels, use a recirculating buffer system.

No bands at all

Check: (1) did you add EtBr or SYBR Safe to the gel or the running buffer? Without a stain you will not see anything. (2) Did the gel run in the right direction? DNA runs toward the red (positive) electrode. (3) Is the PCR actually producing product? Run a positive control.

Frequently Asked Questions

Next Steps

ConductColony — auto-generate gel layouts that match your genotyping batches

ConductColony prints gel-loading maps that match your PCR plate map, so every well is pre-labeled with the animal ID and expected genotype.

Open app →

PCR Master Mix Scaler

Scale the PCR master mix that produces the products you are about to load on this gel.

Browse →

Tissue Digestion Volume Calculator

Scale the lysis buffer volumes for the tissue that feeds the PCR that feeds this gel.

Browse →

Mendelian Ratio & Chi-Square Calculator

After the gel: test whether your observed genotype ratios match expectations.

Browse →

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