Bio-Rad Sub-Cell® Model 192 Cell - Manual (Page 17)

and gels appropriately (Review EtBr Material Safety Data Sheet [MSDS] for proper
disposal methods).
2. Destain the gel for 10-30 minutes in dH
2
O using 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 once 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 wavelength. However, nicking of DNA
will increase below 302 nm. Table 2.5 indicates the percentage of transmittance of UV
light through 1/4
″
(.635 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” (.635 cm)
UV Transparent Plastic
Approximate %
Wavelength (nm) Transmittance
254 0
302 80
360 90
5. Photograph the gel using standard cameras and film (e.g., Bio-Rad Standard Polaroid Gel
Documentation System) or with CCD-based digitized image analysis systems (e.g., Bio-Rad
Gel Doc™ 1000). Gels are generally photographed with a yellow, orange, or red inter-
ference 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.
2.7 Note on Blotting
Nucleic acids within the gel can be transferred to membranes using the techniques of
Southern and Northern blotting. It is beyond the scope of this instruction manual to include
blotting procedures. Consult References 1 and 2 for blotting techniques. Bio-Rad offers a
full-line of nitrocellulose and positively-charged nylon membranes, as well as vacuum and
electrophoretic blotting apparatus for Southern and Northern blotting (Section 6.3).
Section 3
Gel and Electrophoresis Reagents Preparation
3.1 Electrophoresis Buffer Preparation
DNA agarose gel electrophoresis is usually conducted with either Tris-Acetate-EDTA
(TAE) or Tris-Boric Acid-EDTA (TBE). While TAE provides faster electrophoretic migration
of linear DNA and better resolution of supercoiled DNA, TBE buffers have a stronger buffering
capacity for longer- or higher-voltage electrophoresis runs. Bio-Rad offers premixed
50x TAE and 10x TBE buffers for use with the Sub-Cell systems. RNA formaldehyde gels
require a MOPS [3-(N-morpholino)-propanesulfonic acid] electrophoresis buffer.
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