Agarose Gel Electrophoresis Quiz

Agarose Gel Electrophoresis

• Well-separated bands with sharp, distinct edges indicate good resolution in agarose gel electrophoresis.
• Blurry or smudged bands may indicate poor resolution or overloading of the gel.

Use of Agarose Gel Electrophoresis

• Agarose gel electrophoresis can be used to fingerprint gram-negative bacilli.
• It can also be used to characterize silver nanoparticles in industrial products.
• Additionally, it can be used to monitor conformational changes of small, spherical plant viruses.

SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis)

• SDS-PAGE is a discontinuous electrophoretic technique used to separate and identify proteins according to their molecular weight.
• It is widely used in biotechnology, biochemistry, molecular biology, forensic science, and other life science laboratories.

Materials and Reagents Required for SDS-PAGE

• 10X running buffer is required for SDS-PAGE.
• 2X Laemmli loading buffer is also needed for SDS-PAGE.
• Other components required include Tris-HCl, Glycine, SDS, Bromophenol blue, 2-mercaptoethanol, and Glycerol.

Staining and Destaining in SDS-PAGE

• Stains such as Coomassie Brilliant Blue and Silver stain are used to visualize separated proteins in SDS-PAGE.
• De-staining buffers are used to remove excess stain from the gel.

Resolving and Stalking Gel Preparation for SDS-PAGE

• Stalking gel components include Acrylamide, Bis-Acrylamide, Tris-HCL (0.5 M, pH 6.8), SDS, Distilled water, TEMED, and 10% APS.
• Resolving gel components include Acrylamide, Bis-Acrylamide, Tris-HCL (1.5 M, pH 8.8), SDS, Distilled water, TEMED, and 10% APS.

Sample Preparation for SDS-PAGE

• Protein samples are mixed with sample buffer and heated at 95°C for 5 min.
• The mixture is then centrifuged at 16000 xg for 5 min and stored at -20°C or proceeded with gel electrophoresis.

How SDS-PAGE Works

• SDS-PAGE consists of a mixture of DNA fragments of known sizes, typically ranging from a few hundred base pairs to several thousand base pairs.
• DNA markers are run alongside the DNA samples on the gel.

Loading of Samples in Electrophoresis

• Loading dye serves as a visual aid during loading and helps the DNA sink into the wells.
• It also contains tracking dyes that move with the DNA bands during electrophoresis, allowing you to monitor the progress of the run.

Setting Up Electrophoresis Apparatus

• Connect the cables from the power supply to the electrodes in the electrophoresis tank.
• Make sure the positive (anode) and negative (cathode) electrodes are correctly connected.
• Start the power supply to begin the electrophoresis process.

Visualizing the DNA

• The migration of DNA fragments can be visualized by observing the movement of the loading dye or by using a suitable staining method.
• Electrophoresis documentation systems typically use a transillumination source, such as UV light or white light, to illuminate the gel or blot for imaging.