Bio-Rad ReadySub-Cell GT Cells - Manual (Page 13)

Table 2.4 DNA size migration with sample loading dyes
Agarose Concentration (%)
Xylene Cyanol Bromophenol Blue
0.5—1.5 4—5 Kb 400—500 bp
2.0—3.0 ** 750 bp 100 bp
>3.0 *** 125 bp 25 bp
** Sieving agarose such as Certified PCR agarose.
*** Sieving agarose such as Certified low range ultra agarose.
2.4 Nucleic Acid Staining and Visualization
Gels can be removed from the Sub-Cell GT base or gel tray for nucleic acid staining. The gel can also
remain on the UVTP gel tray for staining.
Ethidium bromide staining procedure
1. Place the gel into the appropriate volume of 0.5 µg/ml ethidium bromide (EtBr) stain for 15—30 min.
Use enough staining solution to cover the entire gel.
Caution: Ethidium bromide is a suspected carcinogen and should be handled with extreme care. Always wear
gloves, safety glasses, and a laboratory coat. Dispose of used EtBr solutions and gels appropriately (Review
EtBr Material Safety Data Sheet [MSDS] for proper disposal methods).
2. Destain the gel for 10—30 min in dH
2
O with the same volume used for staining.
Note: Ethidium Bromide can be removed from the DNA with extended destaining. This will cause lower
sensitivity of detection. However, insufficient destaining will create higher background fluorescence.
3. Rinse the gel briefly with dH
2
O to remove any residual staining solution.
4. Place the gel on a UV transilluminator for nucleic acid visualization and analysis. DNA-ethidium bromide
complexes may be illuminated with UV light of 254, 302, or 366 nm. Sensitivity decreases with
illumination at higher wavelengths. However, nicking of DNA will increase below 302 nm. Table 2.5 gives
the percentage of transmittance of UV light through 1/4” (.64 cm) UV-transparent plastic.
Note: Nucleic acids in the gel can be visualized through the UVTP trays. If a UVTP tray is not used,
place household plastic wrap between the UV transilluminator and the gel to avoid contaminating the
transilluminator with nucleic acids or EtBr.
Table 2.5 Percent UV transmittance through 1/4” (.64 cm) UV-transparent plastic (UVTP)
Approximate Wavelength (nm) % Transmittance
254 0
302 80
366 90
5. Photograph the gel using standard cameras and film (e.g., Bio-Rad’s Standard Polaroid Gel
Documentation System) or with CCD-based digitized image analysis systems (e.g., Gel Doc
™ 1000
UV fluorescent gel documentation system). Gels are generally photographed with a yellow, orange, or
red interference filter. Red filters generally give the cleanest background. Bio-Rad offers a full-line of
standard photography and CCD-based imaging systems for nucleic acid gel analysis.
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