* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download الشريحة 1
Molecular cloning wikipedia , lookup
Protein adsorption wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Multi-state modeling of biomolecules wikipedia , lookup
List of types of proteins wikipedia , lookup
Signal transduction wikipedia , lookup
Genomic library wikipedia , lookup
Matrix-assisted laser desorption/ionization wikipedia , lookup
Capillary electrophoresis wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Immunoprecipitation wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Western blot wikipedia , lookup
Biochemistry wikipedia , lookup
Community fingerprinting wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Purposes • • 1. 2. To understand the principle of Gel electrophoresis. To become familiar with the part of the electrophoresis setup. Electrophoresis is a laboratory technique for separating molecules based on their charge and or size. This technique is simple: Biological samples are prepared and placed in gel (matrix). Electricity is then run through the matrix, causing molecules in the samples to separate. There are two components of electrophoresis system: 1. 2. • • • Electrical filed. The physical resistance of the matrix. Electrical filed Many molecules (amino acids, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge. At neutral pH: • • proteins have a unique electrical charged. both DNA and RNA bases are negative charged. Molecules with a negative charge (anions) will be attracted to the positively charged node (anode)……. Red color. Molecules with a positive charge (cations) will be attracted to the negatively charged node (cathode)…… Black color. Separation of a Mixture of Charged Molecules Charged molecules are separated based on their electrical charge and size. Positive Molecules Analyze Charge Separation Size Separation Mixture of Charged Molecules Identify Purify Negative Molecules The physical resistance of the matrix • Electrophoresis is conducted through a gel substances called a matrix. • Matrix acts as a physical barrier to movement of the substances. • This barrier acts as a ”sieve” to separate the molecules by size. • Without any resistance to there movement, “equally charged molecules will migrate through the electrical field at the same rate. • If we put barrier in there way small molecules can move more quickly than large one. Molecule Size: • The molecule suspended in a gel. • The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. • Small molecules can move faster than large molecules. • porous material is made of microscopic particles Porous Material Proteins Entering Porous Material Smallest Move Fastest Components of an Electrophoresis System 1. Power supply and chamber: a source of power supply 2. Agarose gel: a porous material that molecules migrates through 3. Buffer: a fluid mixture of water and ions. 4. Gel casting materials Power Supply Cathode Anode + Buffer Dyes Gel Electrophoresis • They are many types of matrices used in electrophoresis, but all function similarly as physical barrier to movement. Gels can be made from substances such as agarose or polyacrylamide. • Agarose “ a complex sugar chain from red seaweed”. – Non toxic carbohydrate. – It is commonly used in foods (ice cream, and jellies) and many biological mediums. – It has a large pore size good for separating large molecules quickly. • Polyacrylamide “chain of acrylamide molecules”. – It is often used to make plastics and rubber. – It has a small pore size good for separating small molecules. Agarose Gel • A porous material derived from red seaweed • Agarose is highly purified to remove impurities and charge. • Acts as a sieve for separating molecules. • This solid matrix will allow the separation of fragments by size. 1% agarose • Concentration affects molecules migration :• Low conc. = larger pores better resolution of larger DNA fragments • High conc. = smaller pores better resolution of smaller DNA fragments 2% agarose Fragment Resolution Most agarose gels are made with between 0.7% (good separation or resolution of large 5–10kb DNA fragments) and 2% (good resolution for small 0.2–1kb fragments) agarose dissolved in electrophoresis buffer. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case. Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly. 1% gels are common for many applications. % Agarose DNA fragment, kb 0.5 30-1 0.7 12-0.8 1.0 10-0.5 1.2 7-0.4 1.5 3-0.2 • Agarose at room temperature is a 3-dimentional solid matrix. • The smaller the fragments the further the migration or movement through the matrix. small large - Power + Electrophoresis Buffer • TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) – pH buffer. 1. Tris a pH buffer. 2. Acetic acid provide ions to support conductivity and maintain pH. 3. EDTA, prevent brake down of molecules. “all dissolved in water”. The important feature of any buffer is that it contains an electrolyte so that it can conduct electricity. Concentration affects DNA migration • Use of water will produce no migraton • High buffer conc. could melt the agarose gel Purposes for Agarose Gel Electrophoresis • Analysis of molecules size. • Separation and extraction of molecules. • Quantification of molecules. Overview of Agarose Gel Electrophoresis • Gel Preparation • Loading the gel • Running the gel Gel Preparation Agarose is a linear polymer extracted from seaweed. Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer. Melting the Agarose Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven. Loading the gel Running the gel