The document discusses agarose gel electrophoresis, which is used to separate DNA fragments by size. DNA samples are loaded onto an agarose gel and an electric current is applied, causing the negatively charged DNA to migrate through the gel at rates depending on fragment size. Smaller fragments move faster and travel farther than larger fragments. After electrophoresis, DNA bands can be visualized by staining with ethidium bromide and exposing to UV light. Agarose gel electrophoresis is used for applications like analyzing restriction enzyme digestion products and determining DNA sizes.

1. 1
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)
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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
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5. Structure of DNA
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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
 Nucleic acid Samples to be separate
 Micropipettes and Tips
 ddH2O
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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
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D-galactose anhydro-L-galacto-
-pyranose
Red algae
Agarose gel (SEM)

8. Types of Agarose
 Standard Agarose - LE
- Gels at 35-38 oC; Melts at 90-95 oC
- Becomes opaque at high concentrations
 Low Melting Agarose (NuSieve)
- Gels at 35oC; Melts at 65oC
- Often used to isolate DNA fragments from gel
 Intermediate forms/combinations of LE and NuSieve can provide sturdy,
translucent gels at high agarose concentrations
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9. Agarose concentrations for nucleic acid
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% 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
 Available 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.
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Electrophoresis chamber
Electrophoresis chamber Power Pack
+ve
-ve
DNA

12. Buffer
 During electrophoresis water undergoes hydrolysis :
H2O 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 Mg2+ ions which are cofactors for DNA nucleases
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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)
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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 ddH2O
 1x TAE can be made from the stock of 50x TAE and ddH2O

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
C21H20N3Br
Mol. Wt. 394.4

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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".
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18. Sample preparation
 DNA sample 5-10 µL (30-100 ng DNA)+ 6x Gel loading dye (1-2 µL)
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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 ddH2O
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
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Length (cm)

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

21. Method for electrophoresis
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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 log10 of their mol. wt.
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23. End Results
*Small DNA move faster than large DNA
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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 3rd 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 (3rd
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

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