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Questions and Answers
What provides ions to conduct electric current and maintains a stable pH in gel electrophoresis?
What provides ions to conduct electric current and maintains a stable pH in gel electrophoresis?
Which electrode should the DNA fragments migrate towards during gel electrophoresis?
Which electrode should the DNA fragments migrate towards during gel electrophoresis?
What serves as a visual aid during loading in gel electrophoresis?
What serves as a visual aid during loading in gel electrophoresis?
Which system typically uses transillumination sources like UV light for imaging in gel electrophoresis?
Which system typically uses transillumination sources like UV light for imaging in gel electrophoresis?
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What moves with the DNA bands during electrophoresis to help monitor the progress of the run?
What moves with the DNA bands during electrophoresis to help monitor the progress of the run?
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Which component is connected to the electrodes in the electrophoresis tank to start the process?
Which component is connected to the electrodes in the electrophoresis tank to start the process?
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What do well-separated bands with sharp, distinct edges indicate in gel electrophoresis?
What do well-separated bands with sharp, distinct edges indicate in gel electrophoresis?
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What might blurry or smudged bands on a gel electrophoresis image suggest?
What might blurry or smudged bands on a gel electrophoresis image suggest?
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Which technique is used to separate and identify proteins based on their molecular weight?
Which technique is used to separate and identify proteins based on their molecular weight?
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Which type of electrophoresis was developed by Ulrich K. for separating and identifying proteins?
Which type of electrophoresis was developed by Ulrich K. for separating and identifying proteins?
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In which journal article was the evaluation of agarose gel electrophoresis for characterization of silver nanoparticles mentioned?
In which journal article was the evaluation of agarose gel electrophoresis for characterization of silver nanoparticles mentioned?
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What is the primary purpose of using agarose gel electrophoresis?
What is the primary purpose of using agarose gel electrophoresis?
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What is the function of the running buffer in gel electrophoresis?
What is the function of the running buffer in gel electrophoresis?
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Which component is commonly found in the Laemmli loading buffer for gel electrophoresis?
Which component is commonly found in the Laemmli loading buffer for gel electrophoresis?
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What is the function of staining and de-staining buffers in gel electrophoresis?
What is the function of staining and de-staining buffers in gel electrophoresis?
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Which gel preparation step involves components like Acrylamide, Bis-Acrylamide, and Tris-HCL?
Which gel preparation step involves components like Acrylamide, Bis-Acrylamide, and Tris-HCL?
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What should be done after mixing the protein sample with the sample buffer in gel electrophoresis?
What should be done after mixing the protein sample with the sample buffer in gel electrophoresis?
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In gel electrophoresis, what is the primary function of SDS (Sodium Dodecyl Sulfate)?
In gel electrophoresis, what is the primary function of SDS (Sodium Dodecyl Sulfate)?
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Study Notes
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.
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Description
Test your knowledge on agarose gel electrophoresis, a technique used to separate DNA fragments based on their size. Learn to identify good and poor resolution of bands on a gel.
