Gel & blot imaging
A light box that excites the DNA stain in an agarose gel from below, with a camera and emission filter above. UV (302 or 365 nm) excites ethidium bromide and most stains; blue light (≈ 470 nm) excites SYBR-type stains without damaging DNA.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
UV / blue-light transilluminator
A light box that excites the DNA stain in an agarose gel from below, with a camera and emission filter above. UV (302 or 365 nm) excites ethidium bromide and most stains; blue light (≈ 470 nm) excites SYBR-type stains without damaging DNA.
How it differs from the standard method
- Match stain, light and emission filter.
- Expose so the brightest quantified band stays below saturation.
- Use blue light (or brief long-wave UV) when excising bands for cloning.
1A picture that has to be a measurement
Gels and blots end as images, and those images are often quantified: how much PCR product, how much more protein after treatment. A gel documentation system lights a stained gel (UV or blue light) and photographs its fluorescence through an emission filter; a chemiluminescence imager sits a cooled camera in a dark box and integrates the faint light emitted where horseradish peroxidase on a secondary antibody oxidises its substrate.
White is not data
A saturated pixel records only that the camera was full. Imaging software can mark saturated pixels; no quantified band may contain any.
Calibrate in the same image
A mass ladder or a dilution series on the same gel or blot is the standard curve that turns intensity into amount, and shows where linearity ends.
Subtract it locally
Gels glow and membranes have uneven background. Subtract the background next to each band, the same way for every lane.
UV damages DNA
Short-wave UV makes pyrimidine dimers in seconds. If the band is going to be cloned, see it with blue light and a blue-excitable stain, or long-wave UV, briefly.
2Transilluminators and DNA stains
Ethidium bromide fluoresces orange when intercalated in DNA and is excited best by UV (around 300 nm); newer stains such as SYBR Safe or GelRed are less hazardous and SYBR-type stains are excited well by blue light around 470 nm. The emission filter matched to the stain blocks the excitation light; without it the background rises and bands saturate sooner.
3Chemiluminescent westerns
Enhanced chemiluminescence (ECL) is an enzyme reaction, not a dye: light is emitted while substrate lasts and the peroxidase stays active. Over a strong band a high-sensitivity substrate can be consumed in seconds, giving bands with pale centres ('ghost' or 'burnt-out' bands) and a signal that no longer rises with protein.
Key terms
Practise these as flashcards →The range of amounts over which signal is proportional to amount, bounded by noise below and saturation or depletion above.
Pixels at the detector's maximum; their true intensity is unknown.
A DNA ladder with stated amounts per band, used as an intensity standard.
A filter in front of the camera that passes the stain's fluorescence and blocks the excitation light.
Enhanced chemiluminescence: light from peroxidase-catalysed oxidation of luminol-based substrates.
Local exhaustion of ECL substrate over strong bands, so signal stops tracking protein amount.
Correcting lane loading by a stain of all protein in each lane rather than by one housekeeping protein.
Measuring band intensities from an image, usually as background-subtracted integrated volumes.
When to use it
- Documenting and quantifying DNA on agarose gels: product yield, digestion completeness, band ratios.
- Locating and excising a DNA band for cloning with minimal damage: blue-light transillumination.
- Comparing protein amounts between samples on westerns with a CCD imager across a demonstrated linear range.
Compared with alternatives
Measures DNA concentration in solution (fluorometric or UV) more accurately than band intensities.
The blotting and detection that produce the membrane imaged here.
Quantifies a protein over a calibrated standard curve with better precision than a western band.
Separating the DNA that the transilluminator then images.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
A light box that excites the DNA stain in an agarose gel from below, with a camera and emission filter above. UV (302 or 365 nm) excites ethidium bromide and most stains; blue light (≈ 470 nm) excites SYBR-type stains without damaging DNA.
- Match stain, light and emission filter.
- Expose so the brightest quantified band stays below saturation.
- Use blue light (or brief long-wave UV) when excising bands for cloning.