gel electrophoresis one of the latest technology in biotechnology

2. Definition
Electrophoresis is a technique used to separate and
sometimes purify macromolecules - especially
proteins and nucleic acids - that differ in size, charge
or conformation.

3. Purpose of Gel Electrophoresis
A method for separating DNA
Can be used to separate the size of
DNA
RNA
Protein
We will be using it to purify DNA, RNA AND
PROTEINS.

4. Principle of gel electrophoresis:
Gel electrophoresis involves an electrical field.
The molecules to be separated are pushed by an
electrical field through a gel that contains small pores
The molecules travel through the pores in the gel at a
speed that is inversely related to their lengths.
This means that a small DNA molecule will travel a
greater distance through the gel than will a larger DNA
molecule

5. DNA and RNA are negatively charged molecules, they
will be pulled toward the positively charged end of the
gel.
IN case of proteins, first mix it with a detergent called
sodium dodecyl sulfate. This treatment makes the
proteins unfold into a linear shape and 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 electrophoresis
AGAROSE GEL POLYACRYLAMIDE GEL
Poured Horizontally Poured Vertically
Separate large molecules Separate small molecules
Non-toxic neurotoxin
Mostly used for DNA separation Used for DNA or Protein
separation
Staining step: before or after
pouring the gel
Ethidium bromide is mostly
used
Staining step: after pouring the
gel
Coomassie blue stain is mostly
used

7. Gel Electrophoresis of DNA
Materials
Ethidium Bromide (10mg/ml)
Gel Loading Dye, 6X, Blue-Orange (comes with
Promega DNA ladder)
5X TBE (stock)
in a 1L beaker: 850 ml of dH2O, 20 ml of 0.5M EDTA
(pH 8), 54g Tris base stir ,add 27.5 g Boric Acid, stir,
pour into graduated cylinder and fill up to 1L with
dH2O, autoclave

8. 0.5M EDTA (pH 8.0)
46.5 EDTA in 200ml water
5 g of NaOH pellets to adjust pH to 8.0
EDTA won’t dissolve until pH is 8.0

9. Sample preparation
Gel electrophoresis requires some kind of DNA
sample—a plasmid, a PCR product, a segment of a
chromosome, etc
Whatever your sample is, it must be mixed with
loading buffer (containing glycerol and dyes, as
described above) before electrophoresis.
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
1. For one gel, you will need a total of 30 ml of a 1 %
agarose solution.
2. 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 .
3. 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. 4 -Heat the agarose in the microwave on high for 15
seconds.
5-Allow the melted agarose to cool on the bench for two
minutes while you set up the casting tray.
6-Insert a gel plate into a casting tray as shown in Figure.

13. 7-Pour the entire 30 ml of melted agarose into the gel
casting tray. Insert a clean comb (which will form the
wells) near one end, and slide it up against the handles
on the gel plate, as shown in Figure.

15. 8-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 and place it in the gel box.
9-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. The gel is now
ready for use.

16. 10-Use a micropipette and a clean tip to transfer each of
your DNA samples to a well.
11-Place the lid on the gel box and fit the power cords
over the two electrodes.
12-Normally, you would want to stop the gel when the
bromophenol blue (dark blue) dye line is near the
bottom edge of the gel but has not yet started to run
off. Stop it sooner if you need to see small DNA
fragments. Turn off the power supply and disconnect
the leads

17. 13-Carefully remove the gel tray and gel from the gel box.
Don't let the gel slide off! Slide the gel off the tray onto
the UV light box of the photo documentation system

18. GEL ELECTROPHORESIS OF RNA
MATERIALS
Gel Loading Buffer II -
95% Formamide, 18mM EDTA, and 0.025% SDS,
Xylene Cyanol, and Bromophenol Blue.
Gel Loading Solution -
10X solution of 40% Sucrose, 0.17% Xylene Cyanol,
and 0.17% Bromophenol Blue.

19. NorthernMax Formaldehyde Load Dye
NorthernMax-Gly Sample Loading Dye
 An improved formulation used for RNA sample
denaturation in any glyoxal gel protocol

20. 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,
and add enough 1X running buffer to cover the gel by a
few millimeters. Then remove the comb.

21. Prepare the RNA sample.
a. To 1-3 µg RNA, add 0.5-3X volumes Formaldehyde
Load Dye.
To simply check the RNA on a denaturing gel, as little as
0.5X Formaldehyde Load Dye can be used, but to
completely denaturate the RNA, e.g. for Northern blots,
use 3X volumes of Formaldehyde Load Dye.

22. Ethidium bromide can be added to the Formaldehyde
Load Dye at a final concentration of 10 µg/ml. Because
ethidium bromide concentration affects RNA
migration in agarose gels.
Heat denature samples at 65-70°C for 5-15 min

23. 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

24. SDS-PAGE OF PROTEIN
MATERIALS
Stock arcylamide solution
Running gel buffer
Stacking gel buffer
10% ammonium persulfate (APS)
10% SDS
Sample buffer: tris HCL =0.6M Ph= 6.8, 5ml SDS=
0.5g, sucrose= 5g, β- mercapto blue =5ml all are mixed
leaving bromophenol blue.

25. Electrophoresis buffer
Protein stain
De-stain solution

27. PROCEDURE:
Clean the internal surface of the gel plate
Join the gel plate together to form cased
Mix following and transfer it to gel cassede.
Running gel buffer= 8ml
Water= 11.4 ml
Stock acrylamide= 20 ml
10% SDS= 0.4 ml
APS= 0.2 ml
TEMED=14μl

28. Butanol is added after setting the solution
Prepare the stacking gel solution and add 14μl TEMED
and load it into gel cassade after dicarding butanol.
Insert comb to create well for polymerization
Assemble gel cassade in gel electrophoresis tank
Fill the top reservoir with gel electrophoresis buffer
Load the sample
Connect the power supply
continue the electrophoreses until bromophenol
reaches the bottom

29. Dismantle the gel apparatus
Remove the gel and pass it through staining solution .
De-stain the gel to remove excess stain.

30. THANK YOU