Restriction Digestion of Plasmid DNA using Agarose Gel

Electrophoresis

Submitted by
Group 1

Fabunan, Melody Aivi

Gerardo, Mary Antonette
Maguslog, Justine
*Salumbre, Renz
Surquia, Joseph Michael

Submitted to:
Ms. Gardette Valmonte
Ms. Abigail Garcia

October 2, 2007
INTRODUCTION

Plasmids are minute genetic elements that replicate separately from

the chromosome. Majority of plasmids are in the form of double-stranded DNA
(dsDNA) and may either be circular or linear. Plasmid DNA is usually
employed in recombinant DNA technology to clone a specific segment of DNA
resulting most advantageously in that large quantities of these segments can
be prepared. Moreover, plasmid DNA have inherent antibiotic resistance
genes and are used to introduce genes into cells by transformation.

Restriction enzymes, known also as restriction endonucleases,

recognize short DNA sequences which are often palindromic. These enzymes
cleave double-stranded DNA at specific sites within or adjacent to their
recognition sequences. Restriction enzymes have specific requirements
needed for optimal activity of the said enzymes. As such, certain conditions
such as temperature, pH, enzyme cofactors, salt composition and ionic
strength affect enzyme activity and stability.

There exist three types of restriction enzymes: Type I have bipartite

and interrupted recognition sequences; and its restriction activity is usually a
pentameric complex. The cofactors and activators involved in this type Mg2+,
AdoMet and hydrolyzed ATP. The cleavage site is distant and variable from
recognition site. Common examples are EcoK I, EcoA I, EcoB I, CfrA I, StyLT
III and StySP I.

Type II have recognition sequences which are either palindromic or an

interrupted palindrome (giving way for ambiguity). Its subunit structure is a
homodimer and Mg2+ alone is involved as a cofactor. The cleavage site is
defined and within the recognition site, leading to a 3’overhang, 5’ overhang
or blunt end. Common examples are EcoR I, BamH I, Hind III, Kpn I, Not I,
Pst I, Sma I and Xho I.

Lastly, Type III enzymes have non-palindromic recognition sequences

and the cofactors and activators involved are Mg2+, AdoMet, ATP (not
hydrolyzed). The cleavage site cuts approximately 25 bases away from the
recongnition sequence and may not cut to completion. Common Type III
enzymes are EcoP15 I, EcoP I, Hinf III, and StyLT I.

Agarose Gel Electrophoresis is a useful tool for the rapid separation of

DNA fragments. Electrophoresis is usually involved in the monitoring of
enzyme reaction, resolution of DNA fragments for transfer to membranes and
hybridization, or preparation of fragments for labeling etc.

The objectives of this experiment are the following: to electrophorese

the digests on an agarose gel and to determine the sizes of the DNA
fragments generated from digestion of the plasmid pGEX 4T1 with restriction
enzymes.
MATERIALS AND METHODS

The materials utilized in this experiment are plasmid pGEX 4T1;

restriction enzymes which are BamHI, EcoRI, HindIII and Pst I; restriction
enzyme buffers, micropipettors; 0.5-mL microcentrifuge tubes; pipette tips and
crushed ice and Styrofoam cup. For the Agarose Gel Electrophoresis, the
materials are restriction digests; 10X Gel loading dye; 1% Agarose in 1X Tris-
acetate-EDTA buffer; 1X Tris-acetate-EDTA buffer prepared from 40X stock
solution; Ethidium bromide and DNA size markers. The special equipment
used are the microcentrifuge; agarose gel electrophoresis set-up with power
supply, transilluminator and a digital camera.

For the methodolody part of the experiment, eight 0.5-ml microtubes

were prepared for each tube: the first microtube was assigned for the Bam HI,
for the second tube, EcoRI and Sal I, third, Bam HI and EcoRI, fourth, EcoRI,
fifth, BamHI and Sty I, sixth, Sal I, seventh, Sty I and Pst I, and lastly, eighth,
Bam HI, Hind III, and EcoR I. And to each tube, 5 µL of plasmid pGEX-4T-1, 1
µL of appropriate 10X restriction enzyme and 1 µL of appropriate 10X
restriction enzyme were added in sequence. They were then subjected in a
microcentrifuge so that the samples, once settled in the bottom, may be
collected. The tubes were then incubated at 37OC. Consequentially; 4 µl of gel
loading dyes was added to each microtubes. 10 µL from each microtubes
were loaded and subjected to the analysis of restriction fragments thru the
Agarose gel electrophoresis.

For the Agarose gel electrophoresis, the first phase consisted of

preparing the gel. A submarine mini-gel was set up and 1% agarose was
melted in 1X TAE buffer in the microwave oven. The melted agarose was
cooled and poured into the gel-casting tray. The comb was put in and the gel
was allowed to set for 30 minutes. The second phase concerns the
preparation of the samples. For this phase, the digested DNA were taken out
of the refrigerator. 2 µL of gel loading dye was added into each tube. The
contents were mixed and subjected under the microcentrifuge. Once the gel
has set, the gel and tray was placed into the electrophoresis chamber thus
initiating the sample-loading and running of the gel phase. A sufficient amount
of 1X TAE buffer was poured such that the gel was completely submerged
with the wells filled up. 10 µL of each sample were loaded into separate wells
in the gel chamber. 10 µL of the DNA size markers were also loaded in the
first lane. The lid was placed on the chamber and the electrical leads were
connected. The power was turned on and the gel run at about 100 volts.
The last phase of the electrophoresis part is the staining and destaining of the
gel. Once electrophoresis was completed, the gel and tray were removed
from the chamber. The gel was slid into a staining tray containg 200 mL of
distilled water and 2 drops of ethidium bromide. The gel was then stained for
20 minutes and the ethidium bromide was removed and contained in an
amber bottle. 200 mL of distilled water was then added and the gel destained
for 10 minutes. The water used was discarded into a waste bottle. For the
purposes of visualization, the gel was subjected under the UV transilluminator.

RESULTS AND DISCUSSION

 Electrophoresis, in this case Agarose gel electrophoresis, was used to
resolve the mixtures of plasmid DNA fragments, specifically, restriction
enzyme-digested genomic DNA. This mixture contains millions of different
fragments once a restriction enzyme has been applied. Fragments have been
visually identified by use of the UV transilluminator. Furthermore, gel
electrophoresis has been used to purify DNA fragments from other fragments
of various sizes. These fragments are synthesized by restriction enzyme
digestion of the cloned DNA and resolved by agarose gel electrophoresis.

In the field of experimental molecular biology, a supercoiled plasmid

DNA is cut at a single site within multiple cloning sequences. The ability of a
restriction enzyme, according to the Promega Restriction Enzyme Resource,
with the consideration of finding a single site by linear diffusion in the
supoercoiled plasmid is different than for any of the sites in a linear substrate.
Moreover, some enzymes have exhibited great differences in their ability to
cut plasmid DNA. These are usually dependent on buffer conditions.

The assigned restriction enzyme for Group 1 is BamH 1. According to

the Promega website, Bam HI has a minimum of two units necessary to cut
1µg of supercoiled DNA containing a single restriction site. If the Bam H1
cuts, there should be only one fragments and the size remains to be 4969bp.

DNA Ladder

Group 1
Figure 1. Result of Agarose Gel Electrophoresis
 Figure 2. Plasmid Map of pGEX 4T1

REFERENCES
Davis, L.G., W.M. Kuehl & J.F. Battey. Basic methods in molecular
biology (2nd ed.). Appleton & Lange : Norwalk, Connecticut, USA.
1994.

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.

http://www.promega.com

http://www.wikipedia.org