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

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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