Agarose gel electrophoresis

Agarose gel electrophoresis

1. Principle & method of Agarose Gel Electrophoresis Krishnendu Sinha Assistant Professor Department of Zoology Jhargram Raj College

2. 6/9/2020 Krishnendu Sinha_JRC 2 Principles

3. 6/9/2020 3 Krishnendu Sinha_JRC Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g., length in base pairs) for visualization and purification Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode Shorter DNA fragments migrate through the gel more quickly than longer ones Thus, you can determine the approximate length of a DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths) Introduction

4. 6/9/2020 Krishnendu Sinha_JRC 4 If a mixture of electrically charged biomolecules is placed in an electric field of field strength E , they will freely move towards the electrode of opposite charge. However, different molecules will move at quite different and individual rates depending on the physical characteristics of the molecule and on experimental system used. The velocity of movement, ν , of a charged molecule in an electric field depends on variables described by V= Eq / f where f is the frictional coefficient and q is the net charge on the molecule The frictional coefficient describes frictional resistance to mobility and depends on a number of factors such as mass of the molecule, its degree of compactness, buffer viscosity and the porosity of the matrix in which the experiment is performed. Biophysical Principle

5. 6/9/2020 Krishnendu Sinha_JRC 5 DNA or RNA Molecular Weight Voltage Agarose Buffer Visualisation Things that affect the procedure…

6. 6/9/2020 Krishnendu Sinha_JRC 6 Molecular weight

7. 6/9/2020 Krishnendu Sinha_JRC 7 As the voltage applied to a gel is increased, larger fragments migrate proportionally faster those small fragments For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel) Voltage

8. 6/9/2020 Krishnendu Sinha_JRC 8 The most widely used polysaccharide gel matrix nowadays is that formed with agarose This is a polymer composed of a repeating disaccharide unit called agarobiose which consists of galactose and 3,6-anhydrogalactose Agarose gives a more uniform degree of porosity than starch and this may be varied by altering the starting concentration of the suspension (low concentrations give large pores while high concentrations give smaller pores) This gel has found wide spread use especially in the separation of DNA molecules (although it may also be used in some electrophoretic procedures involving protein samples such as immuno-electrophoresis ) Because of the uniform charge distribution in nucleic acids, it is possible accurately to determine DNA molecular masses based on mobility in agarose gels However the limited mechanical stability of agarose, while sufficient to form a stable horizontal gel, compromises the possibilities for post-electrophoretic manipulation Agarose

9. 6/9/2020 Krishnendu Sinha_JRC 9 Gels commonly used in electrophoresis of proteins and nucleic acids Polysaccharide gels are formed by boiling followed by cooling. Rearrangement of hydrogen bonds gives interchain cross linking Agarose is composed of agarbiose Polymerization of acrylamide to form polyacrylamide gel. The polymerization reaction is initiated by persulphate radicals and catalyzed by TEMED. Brief chemistry…

10. 6/9/2020 Krishnendu Sinha_JRC 10 Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE ( Tris -acetate- EDTA) and TBE ( Tris -borate-EDTA ) DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. Electrophoresis Buffer

11. 6/9/2020 Krishnendu Sinha_JRC 11 Loading Dye:

12. 6/9/2020 Krishnendu Sinha_JRC 12 Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility. Visualization

13. 6/9/2020 Krishnendu Sinha_JRC 13 Procedure

14. 6/9/2020 Krishnendu Sinha_JRC 14 •Buffer solution, usually TBE buffer or TAE 1.0x, pH 8.0 • Agarose •An ultraviolet-fluorescent dye, ethidium bromide, (5.25 mg/ml in H2O). Alternative dyes may be used, such as SYBR Green. • Nitrile rubber gloves. Latex gloves do not protect well from ethidium bromide •A color marker dye containing a low molecular weight dye such as " bromophenol blue“ •A gel rack •A "comb“ •Power Supply •UV lamp or UV lightbox or other method to visualize DNA in the gel Equipments

15. 6/9/2020 Krishnendu Sinha_JRC 15 Gel casting tray with comb DNA ladder under three different gel concentration Agarose gel electrophoresis setup

16. 6/9/2020 Krishnendu Sinha_JRC 16 Agarose gel electrophoresis setup Sample loading in agarose gel Partially ran agarose gel with tracking dye front

17. 6/9/2020 Krishnendu Sinha_JRC 17 Add loading buffer to each of your DNA samples. Note:  Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer. Add ethidium bromide ( EtBr ) to a final concentration of approximately 0.2-0.5 μ g/ mL (usually about 2-3 μ l of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. 50x TAE Electrophoresis Buffer Tris free base 242 g Disodium EDTA 18.61 g Glacial Acetic Acid 57.1 ml DDI H2O to 1 l In brief…

18. 6/9/2020 Krishnendu Sinha_JRC 18 Rabindra Reddy, Pulimamidi & Nomula , Raju . (2012). Gel-Electrophoresis and Its Applications. 10.5772/38479. https:// www.addgene.org References

19. 6/9/2020 Krishnendu Sinha_JRC 19 Questions to think about… Can I use PAGE for nucleic acid separation? Can I use agarose for protein separation? What's the upper and lower limit (in MW) for agarose and PAGE? Why we use agarose gel horizontally and why vice-a-vice is true for PAGE? What are the differences of Agarose gel electrophoresis and PAGE?

20. 6/9/2020 Krishnendu Sinha_JRC 20