GEL-DOC SYSTEM

Gel-Documentation System                  

·         Gel Documentation system

·         One of the advanced techniques to visualize nucleic acids and other biomolecules

·         Simple Chemiluminescence phenomenon

·         Use of CCD image and display on computer

·         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.

·         2,7 diamino-10-ethyl 9 phenyl phenanthridium bromide

·         Staining of DNA and RNA, first used in 1970s

·         Highly toxic and carcinogenic

·         Absorbs UV light at 254 nm, 302 nm and 366 nm.

·         Fluorescent emission at 500 nm-590 nm, peak at 590 nm (red)

·         Binds DNA and RNA

·         DNA-EtBr >DNA or EtBr

·         Depends on strandedness od DNA, molecular weight and amount

·         Strongly bound to ds DNA

·         At saturation, one molecule EtBr intercalates at every second base pair in ds DNA

·         Can detect 10 ng DNA

·         At 590nm, 1 ng DNA can be detected

·         Efficiency decreased with ss DNA and RNA

·         Concentration use: 0.5 mg/ml for 30-45 minutes at room temperature

·         Require destaining with MgSO₄ (1.0 mM) for 10-15 minutes

·         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:

·         Photography of stained gels

·         Printout of photographic data

·         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.