Electrophoresis Gel & Dyes

DNA Loading Dye is used to prepare DNA markers and samples for loading on agarose or polyacrylamide gels. It contains two different dyes (bromophenol blue and xylene cyanol FF) for visual tracking of DNA migration during electrophoresis. The presence of glycerol ensures that the DNA in the ladder and sample forms a layer at the bottom of the well. The EDTA included in the solution binds divalent metal ions and inhibits metal-dependent nucleases.

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Bromophenol Blue, Aqueous Dye 30ml Dropper Bottle – MAS 5064

Xylene Cyanol FF , Aqueous Dye 30ml Dropper Bottle – MAS 5065

Ethidium Bromide , Aqueous Dye 30ml in Dropper Bottle – MAS 5066

Tris-acetate-EDTA = TAE buffer 30ml stock Solution – MAS 5073

Tris-acetate-EDTA = TAE buffer 50x stock Solution – MAS 5075

Tris – Boric acid – EDTA = TBE Buffer 50ml stock solution – MAS 5076

Agarose 5gm vial – MAS 5077

D.I Water 5 Liter Can – MAS 5078

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Nucleic acid staining dyes are used for detecting nucleic acids in electrophoresis gels. Historically, the most common dye used for gel staining is ethidium bromide, however due to its toxicity and mutagenicity other dyes that are safer to the user and the environment are preferred.

Adding blue or orange tracking dye to colorless DNA samples allows you to see your sample and obtain information about how DNA molecules move during electrophoresis. Identification is based on the size of DNA bands on the gel after migration of molecules.

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Gel electrophoresis instruments are used to separate nucleic acids and proteins based on their size and charge. Used in forensic, molecular biology, genetics, and microbiology labs, gel electrophoresis instruments are used to run and compare DNA samples.

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Common equipment includes dyes, trays, power supplies, electrodes, cables, gel mixtures, gel dryers, and chemicals such as denaturing agents, gel hardeners, and ampholytes. Selection of an appropriate gel is most important to the electrophoresis process.

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Gel electrophoresis is useful in forensics, biochemistry, genetics, microbiology and other applications requiring analysis of nucleic acid and protein molecule size and characteristics. Protein electrophoresis is also a common method for analyzing blood plasma in medical applications. Electrophoresis often occurs as a preparatory technique prior to cloning, DNA sequencing, Southern blotting, restriction fragment length polymorphism (RFLP), and analysis via mass spectrometry.

Why is bromophenol blue used in gel electrophoresis?

Bromophenol blue is useful as a tracking dye in electrophoresis, an industrial dye, a laboratory acid-base indicator and a biological stain. In gel electrophoresis Bromophenol Blue can be used as a color marker and as as a biological stain. Bromophenol Blue can be used to stain proteins and nucleic acids.

What is agarose gel loading dye?

Loading dye is mixed with DNA samples for use in agarose gel electrophoresis. It generally contains a dye to assess how “fast” your gel is running and a reagent to render your samples denser than the running buffer (so that the samples sink in the well).

How is the agarose gel made?

Agarose is a polysaccharide derivative of agar. Gels are made by heating up agarose in an appropriate buffer. The gel contains microscopic pores that act as a molecular sieve. Certain molecules can also interact with agarose to varying degrees affecting their mobility.

Why agarose gel is prepared in TAE buffer?

The function of TBE and TAE buffer is to allow nucleic acids to move through the agarose matrix. Therefore, the agarose gel must be completely submerged in the buffer. In addition, the TBE or TAE buffer maintains the pH and ion concentration during electrophoresis.

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  • TAE buffer is commonly prepared as a 50× stock solution for laboratory use. A 50× stock solution can be prepared by dissolving 242 g Tris base in water, adding 57.1 ml glacial acetic acid, and 100 ml of 500 mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter.

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TAE buffer is commonly prepared as a 10× stock solution for laboratory use. A 10× stock solution can be prepared by dissolving 48.4 g Tris base in water, adding 12 ml glacial acetic acid, and 20 ml of 500 mM EDTA (pH 8.0) solution, and bringing the final volume up to 200ml.

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TAE and TBE Buffers for Gel Electrophoresis ; 242 g tris base in double-distilled H2O · 57.1 ml glacial acetic acid; 100 ml 0.5 M EDTA solution (pH 8.0).

Tris-acetate-EDTA = TAE buffer is a solution made up of Tris base, acetic acid and EDTA (Tris-acetate-EDTA). It is historically the most common buffer used for 

https://www.austincc.edu/microbugz/handouts/Gel%20Electrophoresis.pdf

using a 0.8% agarose/TAE solution. This means we added 0.4g of agarose to 50mL of. TAE buffer. Then we heated the mixture

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.TBE 5X Stock solution, 54 g of Tris(Trizma), 27.5 grams of boric acid, 20 mL of 0.5 M EDTA solution, Deionized water upto 100ml.

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Ethidium Bromide Solution Preparation and Recipe

  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 10 g of Ethidium bromide to the solution.
  3. Add distilled water until the volume is 1 L.
  4. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved.

PROCEDURE

  1. Step 1: To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg xylene cyanol FF and 1.5 g Ficoll 400. Transfer it to a 15-mL screw-capped graduated tube. Add 7 ml deionized / Milli-Q water. …
  2. Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again. …
  3. 5 ml.
  4. 10 ml.
  5. 25 ml.
  6. 50 ml.
  7. 100 ml.

How is bromophenol blue prepared for gel electrophoresis?

Directions:

  1. Add 25 mg of bromophenol blue to 6.7 ml of ddH2O and mix.
  2. Add 25 mg of xylene cyanol FF and mix.
  3. Add 3.3 ml of glycerol and mix.
  4. Aliquot and freeze at -20 °C for long-term storage.

How do you prepare loading dye for agarose gel electrophoresis?

6x DNA Loading Buffer for agarose gel electrophoresis is typically composed of 30% glycerol (v/v), 0.25% bromophenol blue dye (w/v), and 0.25% xylene cyanol FF dye (w/v)[1]. …

To prepare 5ml of 6x DNA Loading Buffer, combine the following:

• 1.5ml Glycerol.

• 0.0125g bromophenol blue.

Xylene Cyanol FF is used as a tracking dye to monitor the progress of electrophoresis separations. The tracking dye typically migrates with the DNA molecules to around 5 Kb. Xylene Cyanol FF is the tracking dye of choice for monitoring the progress of longer electrophoresis runs.

Xylene cyanol can be used as an electrophoretic color marker, or tracking dye, to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Bromophenol blue and orange G can also be used for this purpose.

To prepare 10 ml of 6X DNA loading dye, weigh out 25 mg xylene cyanol FF. Transfer it to a 15-mL screw-capped graduated tube. Add 7.06 ml of 85% Glycerol and 2.94 ml deionized / Milli-Q water.