UNIVERSITY OF VENDA

DEPARTMENT OF BIOCHEMISTRY

NAME: MADZIVHANDILA LUTENDO

STUDENT NO: 14009226

MODULE: BCM 3622

PRACTICAL 3

09 October 2017

Title: DNA Restriction Digest and Agarose Gel Electrophoresis

AIM

The purpose of the lab was to cut DNA using restriction enzymes, separate DNA fragments
using electrophoretic means. Also, to determine the number of base pairs in fragments of DNA.

HYPOTHESIS

The restriction enzymes will cut the DNA in different places, the smaller fragments will move
faster through the gel.

INTRODUCTION

Restriction endonucleases (or restriction enzymes) are bacterial enzymes that act as defense
mechanisms in these organisms. Restriction endonucleases cleave double-stranded DNA
internally, cutting both strands at regions of specific nucleotide sequences that vary from one
enzyme to another. The sequence cut by a restriction endonuclease is its target site (also called
its recognition site). When foreign DNA, such as viral DNA, is introduced into a bacterial cell,
a restriction endonuclease cuts the foreign DNA into shorter pieces, thereby interrupting most
of the foreign genes. This helps defend the cell against invasion by and expression of genes
that could be harmful to the organism (Karp, 2008). A bacterium protects its own DNA against
digestion by its own restriction enzymes by chemically modifying its DNA soon after DNA
replication, usually by adding methyl groups to bases within the target site of the endonuclease.
The enzyme responsible for protection of the cell’s DNA in this way is a DNA methylase (Davis,
1999).

Restriction enzymes are used in several ways to modify and manipulate DNA molecules. One
common use is to prepare fragments of DNA from one source to be combined with fragments
of DNA from another source – to construct recombinant DNA. Another use is to prepare small
fragments suitable for nucleotide sequence analysis. Another use (which is what you will
perform in today’s lab) is to provide a rough map of the distribution of target sites for different
restriction endonucleases. This is called constructing a restriction map (Prenrki, 2009).

Gel electrophoresis is the most widely used method in molecular biology for separating
macromolecules from one another on the basis of size. It is especially useful for analysing
mixtures of proteins or of nucleic acids with respect to the presence and relative abundance of
molecules of different sizes, and for estimating the size of purified macromolecules; it can also
be used as a step in purification of a specific protein or a specific length-class of DNA (Karp,
2008). Electrophoresis gels for nucleic acids are most commonly prepared with agarose or
with polyacrylamide. Tris-acetate-EDTA (TAE) buffer is a most common buffer solution that
consist a mixture of Tris base, acetic acid, and EDTA. It is used for agarose electrophoresis
analyses of DNA products resulting from PCR amplification, DNA purification protocols, or
DNA cloning experiments, with and without sodium chloride (Bolivar, 2001).

MATERIALS AND METHODS

Conical flask

Cyndrical beaker

Agarose gel

Micro pipettes

Weighing machines

Enzymes (BamHI & HindIII)

Incubator

Ethidium bromide

GROEL

DNA K

TAE buffer

Centrifuge
PROCEDURE

After DNA extraction, Endonuclease restriction digest was conducted using enzymes
purchased from Thermo scientific. BamHI and HindIII fast digest restriction enzyme were
used. The table 2.1 shows how samples were prepared. Confirmation was done using agarose
gel electrophoresis. 0.8% agarose gel was dissolved in 1× TAE buffer (40 mM, 20 mM acetic
acid and 1 mM EDTA) by heating with frequent agitation. The agarose was then cooled to 55
°C prior to addition of ethidium bromide (0.5 ug/ml). The agarose gel was allowed to
polymerise for 15-30 minutes at room temperature. The gel was placed in the electrophoresis
chamber and covered with 1× TAE buffer. Volume of 4 µl of 10× DNA loading buffer (0.25%
bromophenol blue + 30% glycerol) was added to 20 µl of sample ffollowed by loading the
samples at Wells. Electrophoresis was allowed to proceed at 100 volts for 1 hour. The gel was
then visualised using UV light

Table 2.1 Restriction digest

Tube 1 Tube 2 Tube 3 Tube 4

(control/Uncut) (BamHI) (HindIII) (BamHI +
HindIII)
H20 16 µL 15 µL 14 µL 13 µL
Buffer 2 µL 2 µL 2 µL 2 µL
DNA 2 µL 2 µL 2 µL 2 µL
Enzyme - 1 µL 2 µL 3 µL
Total 20 µL 20 µL 20 µL 20 µL
RESULTS

Figure 1: restriction analysis of BamHI and HindIII

0.8% of agarose gel was dissolved in 1× TAE buffer. Ethidium bromide was added as the
fluorescent tag. The agarose gel was run for an hour at 100 volts. The gel was visualised using
UVlight.

DISCUSSION

The hypothesis was that the restriction enzymes will cut the DNA in different places, the
smaller fragments will move faster through the gel, and the control group will not move very
much if at all. Therefore my hypothesis was absolutely correct. I was shown that the restriction
enzymes really do cut the DNA in different locations. I also found out that the smaller
fragments move the farthest containing the least amount of base pairs, especially shown in
“HindIII” of the Gel Electrophoresis picture on page five. The gel concentration must remain
consistent with a steady voltage to avoid fragment migration that is too slow or fast. As well,
if the buffer solution is not of the right composition. A buffer with the wrong pH or ionic
concentration will change the shape of the fragments and their migration times. However I had
some sources of error while doing the lab (Davis, 1999).
CONCLUSION

The restriction enzymes successfully cut the DNA on different places. In this case agarose gel
electrophoresis was used to resolve the mixtures of plasmid DNA fragments, specifically, and
restriction enzyme digested genomic DNA. Fragments have been visually identified by use of
the UV light.
REFERENCES

Bolivar, F. world of genetics (4th Ed.). Construction and Characterization of new cloning
vehicles. Japan.2001.

Davis, L.G., W.M. Kuehl & J.F. Battey. Basic methods in molecular biology (2nd Ed.). Appleton & Lange
: Norwalk, Connecticut, USA.1999.

Department of Biological Sciences. Laboratory Protocols in Cell and Molecular Biology. UST:
Manila. 2007.

Karp, G. Cell and molecular biology: Concepts and experiments. John Wiley & Sons : Asia.
2008

Prenrki, P., F. Karch, S. Iida and J. Meyer. Basic biochemistry (3rd Ed.). The plasmid cloning
vector contains a 482 base pair long inverted duplication. Australia.2009.