2ND YEAR MLT

MOLECULAR BIOLOGY LAB

2010

DNA Gel Electrophoresis

Principle:

Gel electrophoresis is used to separate DNA fragments including PCR products

according to their size and charge.

The product is loaded on the gel that helps DNA filter through itself.

DNA is negatively charged because of the phosphate back bone.

Different sizes of these fragments will cause variation in migration through the gel,
allowing smaller fragments to move faster than the larger ones.

Two types of gels can be used for gel electrophoresis; Agarose gel and acrylamide.

Application of Gel Electrophoresis in Molecular Biology:

1. Estimate DNA molecule size after restriction enzyme digestion (RFLP).
2. Check PCR amplified product.
3. Preparation of DNA to be used in other techniques such as Southern blotting.

Agarose Gel Electrophoresis

It's extracted from seaweed and consists of tiny pores allowing the smaller fragments of DNA to
migrate faster and farther to anode (+ve). Larger fragments face more friction while migrating
through the gel, therefore remaining closer to the cathode (-ve).

Visualization of the gel:

The most common dye used to make DNA bands visible for both type of gel electrophoresis is
Ethidium Bromide (EtBr) dye. It's a highly carcinogenic dye that penetrates Hydrogen bonds of
DNA and fluoresce under UV light.

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MLT

MOLECULAR BIOLOGY LAB

2010

Preparation:

A. Gel Preparation:

1. Select the appropriate gel concentration and prepare it in a flask according to the following table:
Concentrations

0.5%

1%

1.5%

2%

2.5%

3%

Agarose (powder) in
gms

0.5

1.5

2.5

1X TBE Buffer in mls

(liquid)

100

100

100

100

100

100

2. Microwave heated for ~ 1 minute to dissolve the agarose until you get a translucent mixture.

3. Add 5 L of EtBr and swirl to mix.

4. Leave it to cool down at room temperature on the bench for 2 minutes.
5. Pour the gel slowly into the tray. Ensure gel is free from bubbles otherwise push them aside
using a disposable tip.
6. Insert the comb and double check that it is correctly positioned.
7. Allow the gel to set for at least 45 min to 1 hour.
8. Disengage the comb gently.
9. Place the gel in the electrophoresis tank.
10. Pour 1.0x TBE buffer into the gel tank ensuring the gel is completely submerged.

B. Sample Preparation:
1. Pipette 2 l of loading dye into a new tube.
(Loading dye is composed of Bromophenol blue and glycerol. Bromophenol is colored and
helps keep track of the migrating bands and glycerol allows DNA to settle into the wells
avoiding the product to float).
2. Transfer 5l of sample into the tube containing the dye and mix well.
3. Load the sample slowly and carefully into the well.
Prepared by: Ms. Thoraia Shinawi

2ND YEAR MLT

MOLECULAR BIOLOGY LAB

2010

4. Meanwhile load 5l of DNA ladder marker in one of the wells independently.

(DNA marker contains a mixture of DNA molecules of known fragment sizes. After
migration, the unknown sample band is compared to these DNA marker fragments for
size determination).
5. Electrophoresis tank is connected to power supply, making sure the anode and cathode are
correctly connected to ensure proper direction of current flow.
6. The current is adjusted at 100 amperes.
7. Migration of the bands is monitored by visualizing bromophenol blue.
8. The apparatus is switched off and the wires disconnected. The gel is carefully exposed to UV
trans-illuminator. The bands are analyzed and picture taken, while the file is saved in PC.

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MLT

Patient
1

MOLECULAR BIOLOGY LAB

Patient
2

Patient
3

Positive 100 bp

Negative

control

control

Marker

2010

500 bp
400 bp
300 bp
200 bp

100 bp

Polyacrylamide Gel Electrophoresis

Acrylamide (polyacrylamide) Gel:
This is made from the synthetic polymerization of acrylamide. Acrylamide is neurotoxic in
monomeric form however; it is used for high resolution electrophoresis of small molecules.

Prepared by: Ms. Thoraia Shinawi

2ND YEAR MLT

MOLECULAR BIOLOGY LAB

2010

Materials needed for gel preparation:

1. TBE Buffer: To maintain the optimum pH.
2. Acrylamide: Its a monomeric substance that can be activated to form long chain of
polymers (polyacrylamide).
3. Ammonium persulfate (APS): initiates gel formation by activating polymerization of
acrylamide monomers.
4. TEMED: Accelerate the process of polymerization in conjunction with APS.

Buffer
Gel Chamber

A table summarizing differences between Agarose & Polyacrylamide Gel Electrophoresis:

Agarose Gel

Polyacrylamide Gel

Poured Horizontally

Poured Vertically

Separate large molecules

Separate small molecules

Non-toxic

neurotoxic

Mostly used for DNA separation

Used for DNA or Protein separation

Staining step: before or after pouring the gel

Staining step: after pouring the gel

Prepared by: Ms. Thoraia Shinawi