Electrophoresis

On what basis does agarose gel electrophoresis separate molecules?

Agarose gel electrophoresis separates molecules based on size, charge and shape.

soluble

Capable of being dissolved

DNA charge

DNA has a strong negative charge. because of this, it migrates through the gel towards the positive electrode during electrophoresis

is dna soluble?

DNA is soluble in water, but insoluble in alcohol.

Why is glycerol added to the sample solutions before they are loaded into the wells?

Glycerol adds density to the samples so they sink through the buffer and into the wells.

Explain migration according to charge.

Molecules having a negative charge migrate toward the positive electrode; positively charged molecules migrate toward the negative electrode.

why is power applied to the electrophoresis apparatus?

Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). Within a range, the higher the applied voltage, the faster the samples migrate.

do small or large molecules move faster?

Smaller molecules move through the pores faster than larger ones. Molecules can have the same molecular weight and charge but different shapes. Molecules having a more compact shape can move faster through the pores.

purpose of the buffer

The buffer serves as a conductor of electricity and to control the pH.

why did the alcohol layer on top of the dna solution?

alcohol is less dense than water, which is needed to spool the dna

what does cell lysis mean?

breaking open the cell to extract dna

what are nucleotides

building blocks (lego) of dna

how are samples prepared for electrophoresis ?

by mixing them with components that will give the mixture density. These samples can then be loaded with a micro-pipet into wells that were created in the gel by a comb during casting. The dense samples sink through the buffer and remain in the wells.

how are molecules separated in electrophoresis?

charge, size, shape

describe the appearance of isolated dna

clot, mucus

agarose gel

contains microscopic pores which act as a molecular sieve that separates molecules based upon charge, size and shape.

what is the gel made up of?

dissolving agarose powder in boiling buffer solution. The solution is then cooled and poured into a gel tray where it solidifies. The tray is submerged in a buffer-filled electrophoresis apparatus.

what color is the dye?

ethidium bromide or methylene blue.

what is a nucleus

houses dna, largest organelle

staining DNA

important in visualizing the dna using a transfer pipet, place 10 drops of distilled water onto the instastain methylene blue card to liquify the stain. transfer 2-3 drops of the blue liquified stain onto the dna adhering to the spooling rod add 2-3 drops of the liquified blue stain to the beaker from which you spooled the dna. the stain will bind to residual dna in the solution that did not spool onto the rod add 2-3 drops of the liquified stain to a beaker with water. observe how the dye disperses in the water compared to the solution containing dna

DNA spooling

layer alcohol on top of the DNA solution in the beaker. DO NOT MIX THE TWO SOLUTIONS. place tip of pipet on the inside wall of beaker. let alcohol slowly stream down the side twirl the glass rod in a circular motion to spool out dna after spooling 10 times, touch the rod to the bottom of the beaker and twirl it in place to collet any remaining dna

chatode

negative, black end

anode

positive, red end

what are chromosomes

rod-like shapes that contain genes

why is electrophoresis useful?

separates charged biomolecules such as DNA, RNA and proteins. In this process, DNA molecules migrate to the "+" pole as an electrical current passes through the gel. DNA molecules of different lengths separate with the shorter fragments moving faster through the gel compared to the larger fragments. In addition, the "shape" (linear or circular) of the DNA molecule will affect the migration of the DNA molecule.

what properties of dna allow spooling?

soluble in water, not soluble in alcohol

using dye in electrophoresis

using dye samples, the migration of the various colored molecules can be visualized directly in the gel during electrophoresis and do not require staining.

what is more dense: water or alcohol?

water is more dense than alcohol