Agarose gel electrophoresis

i am hafiz m waseem from vehari
bsc in science college multan
msc in university of education lahore pakistan

1. HAFIZ MUHAMMAD WASEEM BA BA G LAHORE

2. Agarose Gel Electrophoresis- Introduction Advanced Analytical Techniques UNIVESITY OF EDUCATION LAHORE

3. Introduction Gel electrophoresis? Agarose is a polysaccharide made from seaweed It is dissolved in buffer, heated and cooled to a gelatinous solid in the form of inert matrix A gel is a colloid, suspension of tiny particles in a medium, occurring in a solid form (like gelatin) Gel electrophoresis refers to the separation of charged molecules like nucleic acids, proteins, etc. when an electric current is applied It is the easiest analyzing macromolecules

4. Introduction How GE works? Frictional force of the gel matrix acts as a molecular sieve which separate the molecules on the basis of their size Macromolecules are forced to move through the pores when electrical current is applied Most agarose gels are made between 0.7% and 2% of agarose in some suitable buffer The rate of migration through the electric field depends on Charge of the molecules Size of the molecules Shape of the molecules

5. Introduction How GE works? After staining, the separated macromolecules in the gel are seen in the form of bands A standard is also run in one lane It is most commonly used technique in biochemistry and molecular biology

6. Introduction Types of GE Polyacrylamide gel electrophoresis (PAGE) is used for separation of proteins ranging in size from 5 to 2,000 kDa It uses sodium dodecyl sulfate (SDS) as detergent which denature the proteins and enable them to move independently of each other Agarose gel electrophoresis uses agarose Pores of an agarose gel are large so it is used to separate macromolecules such as Nucleic acids Large proteins Protein complexes

7. Agarose Gel Electrophoresis -Properties Advanced Analytical Techniques

8. Most agarose gels are made between 0.7% and 3% Low percentage gels are very fragile and are used to separate DNA molecules from 5 to 10 kb in size While high percentage gels are used to separate smaller molecules DNA and RNA molecules (negatively charged) move towards anode when electric current is applied Separation depends upon composition and ionic strength of buffers used Properties of AGE

9. Higher the voltage used, faster the nucleic acids will separate Visualization of the nucleic acids may be achieved by ethidium bromide, silver staining, etc. 3-D structure of agarose gels is held together with hydrogen bonds DNA bands can be cut from the gel and processed further for the research Buffers used contain EDTA to inactivate many nucleases

10. Agarose Gel Electrophoresis -Requirements Advanced Analytical Techniques

11. Requirements of AGE Gel Apparatus A power supply, a gel chamber, combs, micropipettes, ladder, buffers, dyes, UV illuminator, electrodes, cables, gel mixtures, gel doc system, are needed

12. Requirements of AGE Buffers Buffers maintain a pH either by absorbing or releasing Hydrogen ions Buffers - either TBE or TAE provide ions to ensure electrical conductivity along with other functions Not only agarose is dissolved in the buffers but the gel slab is also submerged in the buffers after solidifying TAE has the minimum buffering capacity but has the best resolution as compared with other buffers

13. Requirements of AGE DNA Ladder One of the wells in the gel is loaded with a DNA ladder This is used as a marker to compare the separated DNA fragments in the form of bands in the gel and to estimate their sizes

14. Requirements of AGE Visualizing DNA Ethidium bromide ( EtBr ), a fluorescent dye which is visualized when excited by UV light is generally used Gel is soaked in a solution of EtBr and the DNA bands take up the dye Then the gel is placed under UV illuminator and visualized and photographed for further analysis

15. Agarose Gel Electrophoresis - Procedure Advanced Analytical Techniques

16. Gel is prepared by dissolving the agarose powder as per requirement in the suitable buffer It is heated so that agarose is melted in the buffer The melted agarose is allowed to cool before pouring the solution into a cast A comb is placed in the cast to create wells for loading the samples Place the gel at 4C or at room temperature till it is completely solidified Procedure of AGE Casting of gel

17. Once the gel is solidified, the comb is removed for loading DNA samples Loading buffer is mixed with the samples to increase density and to add some dye ( bromophenol blue) to the samples The gel is placed in electrophoresis unit and is covered with the buffer Load ladder and samples in the wells Electrophoresis is carried at the suitable voltage Procedure of AGE Loading of samples

18. Electrophoresis is carried out horizontally  or vertically Buffer used in the gel is the same as the running buffer in the electrophoretic tank Run the gel at 80-150 volatge until the dye is about 75-80% down the gel Afterwards, the gel is removed from the gel box DNA fragments are visualized on placing the gel under UV source Size of DNA fragments is estimated with the help of ladder Procedure of AGE Electrophoresis

19. Agarose Gel Electrophoresis - Applications Advanced Analytical Techniques

20. It is used to analyze DNA molecules which are cut by restriction enzymes Cut fragments can be used for cloning purposes Analysis of PCR products, e.g. in molecular diagnostics or genetic fingerprinting Separation of DNA fragments for extraction and purification purposes Separation of restricted genomic DNA prior to Southern transfer or of RNA before Northern transfer Applications of AGE

21. DNA molecules can be easily recovered from the gels without any damage It is also used for screening protein abnormalities in various biological fluids like serum, urine, CSF, etc. Size of DNA molecules can be analyzed by lambda DNA ladder Two dimensional electrophoresis is useful for identifying a particular protein from thousands of other proteins between control and treated samples Applications of AGE