Gel electrophoresis is a method used to separate DNA, RNA, and proteins based on size and charge by pushing the molecules through a gel with an electrical current. Smaller molecules move faster and further than larger ones. There are two main types of gels used: agarose gels for larger molecules like DNA and RNA, and polyacrylamide gels for smaller molecules like proteins. The document provides detailed protocols for running gel electrophoresis with DNA, RNA, and the differences between the types of gels and their applications in research and clinical settings.

1. GEL
ELECTROPHORESIS
BY TAUKEER AHME MALIK
UW-19-BIS-MS-008

2. INTRODUCTION
 method for separation and analysis of DNA,
RNA and proteins and their fragments, based
on their size and charge.
 Used in clinical chemistry, Biochemistry,
Molecular Biology etc.

3. Purpose of gel electrophoresis
 used to separate and purify the size of
 DNA
 RNA
 Protein

4. Principle of gel electrophoresis
 Involves an electrical field
 Molecules to be separated are pushed by an
electrical field through a gel that contains small
pores
 Molecules travel through the pores in the gel at
a speed that is inversely related to their
lengths
 Small DNA molecule will travel a greater
distance through the gel than will a larger DNA
molecule

5.  DNA and RNA moves towards the positively
charged end
 IN case of proteins:
 first mix it with sodium dodecyl sulfate
 This treatment makes the proteins unfold into
a linear shape
 Coats them with a negative charge
 which allows them to migrate toward the
positive end of the gel and be separated.

6. Types of gel
 AGAROSE GEL
 Poured Horizontally
 Separate large molecules
 Non-toxic
 Mostly used for DNA or RNA separation
 Staining step: before or after pouring the gel
Ethidium bromide is mostly used

7.  POLYACRYLAMIDE GEL
 Poured Vertically
 Separate small molecules
 Used for DNA or Protein separation
 Staining step: after pouring the gel Coomassie
blue stain is mostly used

8. GEL
ELECTROPHORESIS
OF DNA

9. Sample preparation
 It requires some kind of DNA sample—a
plasmid, a PCR product, a segment of a
chromosome, etc
 sample must be mixed with loading buffer
 Add a volume of loading buffer equal to 1/5 the
volume of your sample and mix it well before
loading your sample on the gel.

10. How to pour and run a gel
 For one gel, you will need a total of 30 ml of a
1 % agarose solution.
 Prepare 300 ml of 10× TBE buffer by
measuring 30 ml into a 500-ml graduated
cylinder, then filling the cylinder to the 300-ml
mark with dH2O.
 Pour 30 ml of the 1× TBE into the flask with
your agarose. The remaining TBE will be your
running buffer, which carries the current from
one electrode to the other.

11.  Heat the agarose in the microwave on high for
15 seconds.
 Allow the melted agarose to cool on the bench
for two minutes while you set up the casting
tray.
 Insert a gel plate into a casting tray
 Pour the entire 30 ml of melted agarose into
the gel casting tray.

12.  Insert a clean comb
(which will form the
wells) near one end,
and slide it up
against the handles
on the gel plate

13.  Let the agarose harden for at least 15 minutes.
 When the agarose has fully hardened,
carefully remove the comb.
 Remove the gel plate, with the gel on it, from
the casting tray
 place it in the gel box

14.  Add running buffer until the gel is completely
submerged
 if you look from the side, it should be covered
by a few millimeters of liquid.
 gel is now ready for use.

15.  Use a micropipette and a clean tip to transfer
each of your DNA samples to a well
 Place the lid on the gel box and fit the power
cords over the two electrodes
 Stop it sooner if you need to see small DNA
fragments.
 Turn off the power supply and disconnect the
leads

16.  Carefully remove
the gel tray and gel
from the gel box
 Slide the gel off the
tray onto the UV
light box of the
photo
documentation
system

17. GEL
ELECTROPHORESIS
OF RNA

18. Protocol
 Prepare the gel.
 Heat 1 g agarose in 72 ml water until
dissolved, then cool to 60°C.
 Add 10 ml 10X running buffer, and 18 ml 37%
formaldehyde (12.3 M).
 Pour the gel using a comb
 assemble the gel in the tank

19.  add enough 1X running buffer to cover the gel
by a few millimeters
 Then remove the comb
 Prepare the RNA sample

20. Electrophoresis
 Load the gel and electrophorese at 5-6 V/cm
until the bromophenol blue (the faster-
migrating dye) has migrated at least 2-3 cm
into the gel, or as far as 2/3 the length of the
gel.
 Results
 Visualize the gel on a UV transilluminator

21. Advantages
 gel is easily poured,
 Does not denature the samples
 The samples can also be recovered
 the resulting gel can be stored in a plastic bag
in refrigerater.

22. Disadvantages
 gels can melt during electrophoresis
 buffer can become exhausted
 different forms of genetic material may run in
unpredictable forms

23. Applications
 DNA sequences can be isolated, analyzed &
cloned
 Protein & DNA analysis
 Determination of impurities
 Molecular biology, Microbiology, Biochemistry
 Use in DNA fingerprinting
 Use in food industry

24. T H A N K
Y O U