Gel-Documentation System
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Gel Documentation system
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One of the advanced techniques to visualize
nucleic acids and other biomolecules
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Simple Chemiluminescence phenomenon
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Use of CCD image and display on computer
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Common imaging method in Molecular Biology
lab
Principle:
Principle of fluorescence With
fluorescent staining of nucleic acids, a fluorescent substance that has bound
to nucleic acids is excited by ultraviolet irradiation and emits fluorescent
light. The fluorescent substance Ethidium Bromide binds specifically to nucleic
acid and the amount of bonding depends on the molecular weight and
concentration of the nucleic acid. In other words, a band for a large molecular
weight or large amount will shine brighter; conversely, fluorescence will be
weaker for a band for a small molecular weight or small amount.
Instrumentation:
Figure 1: Picture of Gel-Documentation System
Optical diagram is not shown here:
Different parts in
Gel-Doc System:
1. Source of irradiation:
UV
Transilluminator 20 x 20 cm, 312 nm (254 nm selectable)
2. Base Plate
A
sample tray, a gel viewer
3. A set of filters
4. Imaging device
A
camera (CCD with high resolution) unit integrated with darkroom, camera controller,
video monitor and printer.
5. Readout system
Computerized system
Samples stained
with:
1. Fluorochrome-stained
samples requiring UV excitation. E.g., EtBr, SYBR Green, SYPRO Orange, SYPRO
Ruby.
2. Samples
having bands stained, using a white transilluminator. E.g., CBB, silver stain,
X- ray film (without staining).
3. Samples
having membrane or TLC coloring using in-cabinet lamp. E.g., antigen-antibody
reactions, various coloring reactions
For
example: - Fluorescent membrane, fluorescent TLC
(epi UV (AE-6932FXCF, option))
-EtBr/SYBR Green fluorescent gel (translucent
UV)
-CBB/Silver staining gel (using Gel Viewer)
-Color-producing membrane/color-producing TLC
(interior lamp)
-Other visualizable samples*EtBr: Etidium
bromide *SYBR is registrated trademark of Molecular Probes, Inc.
In
many cases: Ethidium bromide is used.
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2,7 diamino-10-ethyl 9 phenyl phenanthridium
bromide
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Staining of DNA and RNA, first used in 1970s
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Highly toxic and carcinogenic
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Absorbs UV light at 254 nm, 302 nm and 366
nm.
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Fluorescent emission at 500 nm-590 nm, peak
at 590 nm (red)
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Binds DNA and RNA
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DNA-EtBr >DNA or EtBr
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Depends on strandedness od DNA, molecular
weight and amount
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Strongly bound to ds DNA
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At saturation, one molecule EtBr intercalates
at every second base pair in ds DNA
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Can detect 10 ng DNA
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At 590nm, 1 ng DNA can be detected
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Efficiency decreased with ss DNA and RNA
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Concentration use: 0.5 mg/ml
for 30-45 minutes at room temperature
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Require destaining with MgSO4 (1.0
mM) for 10-15 minutes
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At 0.1 mg/ml EtBr
concentration no destaining requires.
Problems:
With continued irradiation of ultraviolet rays, the fluorescence of a
band gradually weakens. This is particularly striking when the molecular weight
or the amount of the sample is small. It even disappears in 20-30 seconds to about
1 minute due to ultraviolet irradiation at 254 nm (a short wavelength). The
ATTO Printgraph prevents excess irradiation and can minimize ultraviolet
irradiation time by pressing FREEZE button once you get a sufficient band
image.
Figure 2: DNA bands observed under Gel-Doc System
Purpose and Analysis:
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Photography of stained
gels
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Printout of photographic
data
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Saving of photographic
data
Image
data is displayed in real-time. Images still displayed can be printed out with
a video printer or saved to a Compact Flash media.
Note: Before throwing gel containing EtBr, it must be
autoclaved to damage its structure to neutral.
Preserve foods by using salts and sugars
works by
