What causes DNA smearing during electrophoresis?
1. Improperly prepared gel: If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear. If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel.
What is undigested DNA in gel electrophoresis?
Here in gel electrophoresis, digested refers to the DNA broken into pieces by Restriction Endonuclease. While undigested DNA means the DNA on which the restriction endonuclease has not acted and the DNA is unbroken.
How does DNA separate in gel electrophoresis?
Gel electrophoresis is a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. DNA fragments are negatively charged, so they move towards the positive electrode.
Which way does DNA migrate in gel electrophoresis?
negatively
Gel electrophoresis and DNA DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.
What does the smearing indicate?
Smearing also results from poor sample quality. For example, a DNA sample contaminated with protein or containing too much salt may produce smearing. Degraded or denatured samples also yield poor results, including smeared bands.
Why do gels smear?
Why do we run and undigested DNA sample?
We ran one sample of undigested DNA on the gel so that we could compare it (in size) to the digested samples. The undigested DNA ran faster than the digested sample because it is supercoiled, so it is more compact and runs through the gel like a smaller piece of DNA. You just studied 21 terms!
How does undigested plasmid run on gel?
Undigested plasmid may have two forms show up in its lane: CCC dimer and CCC monomer forms. The dimer forms, due to their larger and doubling size compared to monomers, usually move slower than the monomers. Therefore, it will appear higher in a gel than a monomer.
How is DNA concentration calculated in gel electrophoresis?
DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA.
How is gel prepared for DNA electrophoresis?
1. Preparation of the Gel
- Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution.
- Add running buffer to the agarose-containing flask. Swirl to mix.
- Melt the agarose/buffer mixture.
- Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml.
Why do DNA fragments migrate during electrophoresis?
Gel electrophoresis uses electricity to separate fragments of DNA based on their length. The negative charge on the sugar-phosphate backbone of DNA polymers cause them to migrate towards the positive electrode when placed in an electrical field.