Gel electrophoresis practical

Practical of agarose gel electrophoresis in detail, description, chemicals, and performed experimet

1. 1 Agarose Gel Electrophoresis Dr. Govinda Navale Dept. of Chemistry, IIT-Roorkee Course CYN-532

2. Why electrophoresis ? To separate DNA/protein fragments from each other To determine the sizes of DNA/proteins fragments To determine the presence or amount of DNA/protein To analyse the restriction digestion products Agarose (for DNA/RNA), PAGE (for Proteins) 2

3. Agarose Gel Electrophoresis Separates DNA or RNA molecules by size, charge and shape Achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis) Shorter molecules move faster and migrate faster than longer ones Separation depends on how the sample and gel (%) are prepared 3

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5. Structure of DNA 5 S S S S P P P P 5’ 3’ 5’ 3’

6. Materials to be required: Agarose Gel casting tray and combs Electrophoresis chamber and power pack Buffer (1x TAE/TBE) Staining agent (dye) DNA ladder N ucleic acid Samples to be separate Micropipettes and Tips ddH 2 O 6

7. Agarose A linear carbohydrate polymer (polysaccharide) extracted from seaweed algae Agarobiose forms a porous matrix as it gels – shifts from random coil in solution to structure in which chains are bundled into double helices 7 D-galactose anhydro -L- galacto - -pyranose Red algae Agarose gel (SEM)

8. Types of Agarose Standard Agarose - LE - Gels at 35-38 o C ; Melts at 90-95 o C - Becomes opaque at high concentrations Low Melting Agarose ( NuSieve ) - Gels at 35 o C ; Melts at 65 o C - Often used to isolate DNA fragments from gel Intermediate forms/combinations of LE and NuSieve can provide sturdy , translucent gels at high agarose concentrations 8

9. Agarose concentrations for nucleic acid 9 % Agarose (w/v) Size Range (base pairs) • 0.5 200-30,000 • 0.75 700-20,000 • 1.0 500-10,000 • 1.5 200-3000 • 2.0 100-2000 • 3.0 (Nu-Sieve) 70-1500 • 4.0 (N-S) 40-900 • 5.0 (N-S) 30-600

10. Gel Casting Trays and Combs A vailable in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape/ rubber stopper A comb is placed in the liquid agarose after it has been pour. 10

11. 11 Electrophoresis chamber Electrophoresis chamber Power Pack + ve - ve DNA

12. Buffer During electrophoresis water undergoes hydrolysis : H 2 O H + + OH - Buffers prevent the pH from changing by reacting with the H+ or OH- products Components of buffer ( TBE or TAE) used : - TRIS [ tris ( hydroxymethyl ) aminomethane ] - Boric Acid or acetic acid - EDTA ( Ethylenediamine tetra-acetic acid ) - for chelating the Mg 2+ ions which are cofactors for DNA nucleases 12 Tris is a strong base and borate/AA is an acid, combination of both maintains the pH nearly 8 to 8.5

13. Tris Buffer Preparation (50x and 1x) 13 Sr. No. Chemicals Mol. Wt. Main stock (50x) 50x ( grms /L) 1x working 1x ( grms /L) 1 Tris base 121.1 g/l 2 M 242.2 g/l 40 mM 4.844 g/l 2 acetic acid / 57.1 ml/l 1 M 57.1 ml/l 20 mM 1.21 ml/l Boric acid 61.84 g/l 4.4 M 275 g/l 88 mM 5.5 g/l 3 EDTA 372.24 g/l 50 mM 18.612 g/l 1 mM 0.372 g/l Adjust the volume by adding ddH 2 O 1x TAE can be made from the stock of 50x TAE and ddH 2 O

14. Staining of DNA To make DNA fragments visible after electrophoresis , the DNA must be stained The favourite— ethidium bromide (EtBr) When bound to DNA it fluoresces under ultraviolet light (reddish –orange colour) Convenient because it can be added directly to the gel Sensitive—detects 0.01ug of DNA Cons: EtBr is mutagenic (care should be taken) Other Dyes: Methylene blue: syber safe; xylene cyanol ; bromophenol blue, Gel red dye 14

15. Ethidium bromide EtBr is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. Inserting itself between the base pairs in the double helix UV absorbance maxima at 300 and 360 nm and emission maxima at 590 nm . Detection limit of bound DNA is 0.5-5 ng/band. It is mutagenic so care must be taken while handling the dye . The standard conc. used in staining DNA : 0.5-1ug/mL 15 C 21 H 20 N 3 Br Mol. Wt. 394.4

16. 16 Contd …

17. DNA ladder It is a solution of DNA molecules of different length DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample . The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a " ladder ". 17

18. Sample preparation DNA sample 5-10 µL (30-100 ng DNA)+ 6x Gel loading dye (1-2 µL) 18 Gel loading dye (6X, 10 mL) 25 mg bromophenol blue (0.25 %) 25 mg xylene cyanol FF (0.25 %) 3.3 ml glycerol (30 %) 6.7 ml ddH 2 O Other dyes combinations Ficoll & Orange G Sucrose & xylene cyanol / bromophenol blue  Glycerol & bromophenol blue Micropipettes

19. Applied voltage ↑ voltage, ↑ rate of migration The higher the voltage, the more quickly the gel runs But if voltage is too high, gel melts The best separation will apply voltage at no more than 5V/cm of length 19 Length (cm)

20. UV Transilluminator Platform for Gel UV protecting glass W avelength for flexibility and convenience: 254/312/365 nm

21. Method for electrophoresis 21

22. DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size. Linear DNA migrate inversely proportional to the log 10 of their mol. wt. 22

23. End Results * Small DNA move faster than large DNA 23 Ladder S1 S2 S3 S4 S5

24. Experimental Check Video at: https ://www.youtube.com/watch?v=_ AhL2jKo3tI

25. References Sambrook J, Russel DW (2001). Molecular Cloning: A Laboratory Manual 3 rd Ed. Cold Spring Harbor Laboratory Press . Cold Spring Harbor , NY. Leonard G. D, and James F. B. Basic Methods in Molecular Biology, 1986 Joseph Sambrook ; David Russell. "Chapter 5, protocol 1". Molecular Cloning - A Laboratory Manual. 1 (3 rd ed.). p. 5.4. ISBN 978-0-87969-577-4 Zimm BH, Levene SD (May 1992). "Problems and prospects in the theory of gel electrophoresis of DNA“. Quarterly Reviews of Biophysics . 25 (2): 171–204 Jean-Louis Viovy (2000). "Electrophoresis of DNA and other polyelectrolytes: Physical mechanisms". Reviews of Modern Physics. 72 (3): 813–872. Bibcode:2000RvMP...72.. 813V https :// en.wikipedia.org/wiki/Agarose_gel_electrophoresis https ://www.slideshare.net/harshit172/agarose-gel-electrophoresis-25523393

26. Thank You Questions ?? 26