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Gel Electrophoresis, Imaging and Analysis

This section deals with agarose and polyacrylamide gel electrophoresis associated with Nucleic acid (NA) workflows. Gel electrophoresis is a method of molecular sieving of DNA or RNA on a matrix to asses size and quality of NA.

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Typical applications include:

  1. Quality of Nucleic acid after isolation
  2. Visualizing product after end-point PCR
  3. Southern or Northern blotting
  4. Electrophoretic mobility shift assays (for NA-protein complexes), also called gel mobility shift assays

Gel electrophoresis can be native or denaturing, depending on the use of denaturing agents in the running buffer. Typically non-denaturing gels use 1x TAE (Tris-acetate EDTA) while denaturing gels are run with 1x TBE (Tris borate EDTA). These can be run on agarose or polyacrylamide gel electrophoresis (PAGE) as the sieving matrix.

Agarose gels have lower resolving power than PAGE, and are used for visualizing NA of large size ranges (50-20k bp) vs. 5-500 bases for PAGE. Polyacrylamide is preferred for single stranded DNA analysis. Comparisons of the native and denaturing methods using agarose and PAGE follow.

Agarose gels: Native and Denaturing Gels

Gel electrophoresis apparatus & combsGel electrophoresis apparatus & combs
Agarose gel stained with SYBR safe stainAgarose gel stained with SYBR safe stain

Native Agarose Gels Denaturing Agarose Gels
Applications such as EMSA study nucleic acid- ligand complex formation. For such complex assessment it is critical that the NA is not denatured. It is also used for serum protein analysis in diagnostics Employed for most applications: end-point PCR product assessment, Cloning, DNA size and quality assessment, Northern analysis, Southern analysis.
RNA quality can be assessed using non-denaturing (Native) Agarose gels. In these cases the RNA or DNA size markers are run alongside total RNA and the quality of the ribosomal bands 16S &23S (prokaryotes) or 18s & 28s (eukaryotes) is evaluated. Genomic DNA quality is assessed by running on denaturing gels. Typically high quality genomic DNA runs at very high molecular weight (several kb). Denatured RNA is seen as a smear of low molecular weights.
For applications such as EMSA, the gel loading dyes do not contain Ethidium bromide or SYBR stains, as these can impact complex formation by inter-chelating with the Nucleic acid. Such gels are stained after electrophoresis to visualize the complex. Can be run with the NA pre-mixed with gel visualization dyes such as Ethidium bromide, SYBR gold.
Reagents Reagents
25X TAE Ultrapure DNA typing Grade (50 TAE buffer) TBE buffers DNA size ladders DNA size ladders
Gel loading solution (All purpose native agarose) SYBR Safe Stain with 1X TAE RNA Gel Loading Dye (2X) RNA size ladders  
Common reagents
Agarose, Agarose Tablets, Low melting agarose for Cloning Gel electrophoresis apparatus, combs and consumables Ethidium bromide
Quick TipsUse SYBR Safe dye solutions to prevent work with mutagenic Ethidium bromide. These have same sensitivity as EtBR, can be visualized using non-UV light sources such as Blue light (500nM).

Polyacrylamide gels: Native and Denaturing Gels

Native Polyacrylamide gels Denaturing Polyacrylamide gels
Applications such as EMSA study nucleic acid- ligand complex formation. For such complex assessment it is critical that the NA is not denatured, DNA- pulled down assays and others. More information Typically used for Ribonuclease protection assays S1 nuclease protection assays and DNAse I footprinting assays, DNA or RNA sequencing Protocol
For applications such as EMSA, the gel loading dyes do not contain Ethidium bromide or SYBR stains, as these can impact complex formation by inter-chelating with the Nucleic acid. Such gels are stained after electrophoresis to visualize the complex. Nucleic acid molecules in such experiments are visualized using dyes such as toluidine blue, SYBR green, and ethidium bromide. Nucleic acids can also be tagged with radio isotopes such as P32 or S35, or fluorescent dyes. (Reference)
Reagents Reagents
25X TAE TBE buffers DNA size ladders
Ultrapure DNA typing Grade (50 TAE buffer) Gel Loading Buffer II (Denaturing PAGE) RNA size ladders
Common reagents
TEMED Ammonium per sulfate (APS) Ultra pure Acrylamide Bis-acrylamide Acrylamide/Bis 19:1 is a 40% (w/v) solution of acrylamide (38%) and bis-acrylamide (2%) 
Quick TipsUse SYBR Safe dye solutions to prevent work with mutagenic Ethidium bromide. These have same sensitivity as EtBR, can be visualized using non-UV light sources such as Blue light (500nM).

Gel Imaging Instruments: Basic Gel doc systems

Application InGenius G:Box NuGenius
 
Fluorescence UV transilluminator Yes Yes Yes
Fluorescence UV epi   Yes  
Blue LIght Yes Yes Yes
Visible Stain converter Yes Yes Yes
Colony Plate Yes Yes Yes
Spot Blot Yes Yes  
Thin Layer Chromatography (TLC)   Yes  

Blue light transilluminators from Syngene use 470 nm LED arrays for illumination. UltraSlim LED model can be side mounted on systems such as the In: Genius 3 or U: Genius 3 systems and the UltraBright LED can be mounted on the G:BOX series. These blue light are a safe alternative to the use of harmful UV transilluminators. Such systems prevent damage to the nucleic acid due to harmful UV. As seen in the table, a wide range of safe dyes can be used with these transilluminators.

Systems   Compatible Dyes
UltraSlimLED transilluminators Coomassie Fluor Orange, Ethidium bromide, Gel Red, Gel Green, SYBR Safe, SYPRO Ruby, SYPRO Orange, SYBR Green I & II, UltraSafe Blue
UltraBright LED transilluminators Coomassie Fluor Orange, Ethidium bromide, Gel Red, Gel Green, SYBR Safe, SYPRO Ruby, SYPRO Orange, SYBR Green I & II, UltraSafe Blue

Gel Imaging Instruments: Advanced Gel doc systems

G:Box Chemi XX6G:Box Chemi XX6
GeneGnome XRQGeneGnome XRQ
Application GeneGnome XRQ G:Box Chemi XRQ G:Box Chemi XX6 G:Box Chemi XX9 G:Box Mini
Fluorescence UV transilluminator   Possible option Possible option Possible option Possible option
Fluorescence UV epi   Possible option Possible option Possible option  
Blue LIght   Possible option Possible option Possible option Possible option
Visible Stain converter   Possible option Possible option Possible option Possible option
Colony Plate   Possible option Possible option Possible option Possible option
Spot Blot   Possible option Possible option Possible option Possible option
Fluorescence Color   Possible option Possible option Possible option Possible option
Chemiluminescence Yes Yes Yes Yes Yes
Fluorescence IR     Possible option Possible option Possible option
Proteomics (2D)     Possible option Possible option Possible option

General equipment

Common Equipment

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