HUMAN GENETICS LAB

Lab 7: Gel electrophoresis of PCR products

1. OBJECTIVES OF LAB EXERCISE:

To perform gel electrophoresis of PCR products

To determine the presence or absence of PCR products and quantify the size (length of
the DNA molecule) of the amplicons.

2. GEL ELECTROPHORESIS

Electrophoresis is a method of separating substances based on the rate of movement while under
the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose
gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes
clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-
like slab. During electrophoresis, the gel is submersed in a chamber containing a buffer solution
and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the
gel by the electrical current. Under an electrical field, DNA will move to the positive electrode
(red) and away from the negative electrode (black). Several factors influence how fast the DNA
moves, including; the strength of the electrical field, the concentration of agarose in the gel and
most importantly, the size of the DNA molecules. Smaller DNA molecules move through the
agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA
will be visualized by the use of a dye that binds to DNA.
Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and
yield. PCR products can range up to 10kb in length, but the majority of amplifications are at 1kb
and below. Electrophoresis reveals the size of the product band, which is compared with the
predicted result. Electrophoresis also shows how much of this band was produced, and reveals the
presence or absence of any unintended amplification products.

2.1. Factors affecting movement of molecules in gel electrophoresis

Agarose concentration: By using gels with different concentrations of agarose, one can resolve
different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small
DNAs, while low agarose concentrations allow resolution of larger DNAs. (Figure 1.)
 Figure 1. Separation of DNA on gel of different concentration

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of
DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-
borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due
to differences in ionic strength. Buffers not only establish a pH, but provide ions to support
conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration
of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough
heat may be generated in the gel to melt it.

2.2. DNA Ladder

In order to be able to determine the size of the separated fragments, DNA ladder should be used.
It comprises fragments of specific base pair lengths for estimation of molecular size of the
fragments. For easy determination of fragment size by visual reference, several DNA ladders
include bands that are two to three times more intense than the other bands. In this lab, we will use
HindIII-digested Lambda DNA as a molecular weight marker (Figure 2).

Lambda is a temperate Escherichia coli bacteriophage. The virion DNA is linear and double-
stranded (48,502 nt). Phage lambda DNA is a common substrate for restriction endonucleases and
for generating DNA size marker fragments. The HindIII digest of lambda DNA yields 8 fragments
suitable for use as molecular weight standards for agarose gel electrophoresis.
 Figure 2: HindIII-digested lambda DNA used as molecular weight standard for agarose gel
electrophoresis.

2.3. Gel loading Dye

DNA Loading Dye is used to prepare DNA markers and samples for loading on agarose or
polyacrylamide gels. It contains bromophenol blue for visual tracking of DNA migration during
electrophoresis. The presence of glycerol ensures that the DNA in the ladder and sample forms a
layer at the bottom of the well. The EDTA included in the solution binds divalent metal ions and
inhibits metal-dependent nucleases.

2.4. Nucleic acid stains

Ethidium bromide is an intercalating agent commonly used for detection of nucleic acids, in
particular double stranded DNA. Although a highly sensitive stain, ethidium bromide is
notoriously unsafe. Not only is it a very strong mutagen, it may also be a carcinogen or teratogenic.
It is harmful if swallowed and very toxic by inhalation, as well as being irritating to the eyes,
respiratory system and skin. Additionally it carries the risk of irreversible effects. Ethidium
bromide can therefore pose a major safety problem for the researcher and be an environmental
hazard during disposal.
A number of safer nucleic acid labels suitable for use in electrophoresis are available on the market
like SYBRSafe, GelRed, GelGreen and EvaGreen.
On todays lab we will be using Fast Blast DNA stain.
Fast Blast DNA stain is a convenient, safe, and nontoxic alternative to ethidium bromide for the
detection of DNA in agarose gels following electrophoresis. Fast Blast contains positively charged
dye molecules that are attracted to and bind to the negatively charged phosphate groups on DNA
molecules. The proprietary dye formula stains DNA deep blue in agarose gels and provides vivid,
consistent results. Fast Blast DNA stain is provided as a 500x concentrate that must be diluted
prior to use. The stain can be used as a quick stain when diluted to 100x or as an overnight stain
when diluted to 1x. Because Fast Blast is a non-fluorescent visible stain, the staining protocol takes
longer than traditional fluorescent DNA stains that give rapid results. The sensitivity of Fast Blast
DNA stain is 50 ng of DNA resolved in an agarose gel.
Although the stain is nontoxic, latex or vinyl gloves should be worn while handling the stain or
stained gels to keep hands from becoming stained blue. Lab coats or other protective clothing
should be worn to avoid staining clothes.

3. PROCEDURE

Chemicals:

Agarose
HindIII-digested lambda DNA
1x Fast Blast DNA Stain
Distilled water
1x TBE buffer (for the gel preparation)
0.5x TBE buffer (running buffer)
DNA samples
6x DNA Loading Dye.

Lab Equipment:

Graduated cylinder
Balance
Erlenmeyer flask
Microwave oven
Micropipettes and tips
Test tubes
Test tube rack
Electrophoresis apparatus.
 Protocol:

1. Mix 0.4 g of agarose with 40 ml of 1x TBE buffer.

2. Heat the mixture in the microwave oven until agarose gets completely dissolved.
3. Pour the mixture into the tray and let it solidify. Try to avoid the formation of bubbles. (Do
not forget to put the comb before pouring the gel.)
4. In the meantime, prepare DNA samples for running on the gel. Mix 10 l of DNA sample
and 2 l of 6x DNA Loading Dye in the test tube. Keep samples on ice before loading.
5. Prepare DNA ladder in a separate tube using 1 l of HindIII-digested lambda DNA, 6.5 l
of TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA), and 1.5 l of 6x DNA
Loading Dye. Incubate this sample at 60C for 3 minutes and immediately put on ice until
loaded on the gel.
6. When the gel gets solidified, carefully remove the comb.
7. Add 1x TBE buffer (running buffer) until the gel is completely immersed into it.
8. Run the DNA ladder in the first lane and DNA samples in the following lanes using 10 l
samples.
9. Turn on the electrophoresis apparatus and run the gel for 45 minutes on 110 V (constant)
and 150 mA.
10. Stain the gel by covering it with 1x Fast Blast DNA Stain and shaking it gently at room
temperature overnight.
11. Analyze your gel under the visible light or using GelDoc machine.