Student name: Nguyn Th Minh Hi

ID: USTH BI4 059

Group: 3

Practical report
Restriction nucleases and Gel
electrophoresis

A. INTRODUCTION
1. Purpose
In this lab we will practice to cut circular DNA (a plasmid) by restriction enzyme
and finding the movement of each sample plasmid using agarose gel
electrophoresis.

2. Background
Plasmids are small (1000-50,000 bp), circular piece of DNA found in many
organisms, but are distinct from the organisms own genome. While most are
found in bacteria (about 5% of the DNA that occurs in a bacterium is plasmid
DNA), others can be found in yeast, plants and animal. There are different
plasmids, but most certain features in common. All have an origin of replication
(the cell copies the plasmid DNA before every cell division). This ensures that
both daughter cells receive a copy of the plasmid. So, once of the strain is
inflected, all of its descendants will also be inflected. Plasmids may also carry from
one to a dozen genes that can code for a variety of traits. The commonest type of
gene provides the host with resistance to a specific antibiotic.
Restriction endonucleases are bacterial proteins that cut DNA at certain
sequences calls restriction sites. The first restriction enzyme was isolated in 1970.
Since that time, biochemists discovered hundreds of these enzymes which all
cleave different DNA sequences.
Gel electrophoresis is a method for separation and analysis of macromolecule and
their fragments, based on their size and charge. The most comment gel is agarose
which is a polysaccharide derived from seaweed that will dissolve in liquid when
boiled and then solidify to form a gel when it cools.
Ethidium bromide is the most commonly used dye for DNA and RNA detection
in gels. Ethidium bromide is a DNA intercalator, inserting itself between the base
pairs in the double helix. Ethidium bromide has UV absorbance maxima at 300 and
360 nm, and an emission maximum at 590 nm. The detection limit of DNA bound
to ethidium bromide is 0.5 to 5.0 ng/band.

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B. MATERIALS AND METHODS

1. Materials
Restriction enzymes:
BamHI and XhoI, 10X Buffer TangoTM, Sterile distilled water
(molecular grade)
Regents

Tris-acetate-EDTA (TAE) buffer ( for electrophoresis)

Stock solution 50X TAE (for 500ml):
121g Trisbase in 250 ml bidistilled water (ddH2O), stir to dissolve
Add 28.6ml acetic acid
Add 50ml 0.5M EDTA pH 8.0
Add ddH2O to 500ml
Working solution 1X TAE: The working solution of 1X TAE buffer
is made by simply diluting the stock solution by 50X in deionized
water.
Other reagents:
Agarose
Loading buffer (loading dye)
DNA ladder (DNA maker)
Ethidium bromide.

Equipment

Water bath
Electrophoresis apparatus (the electrophoresis chamber, casting tray
and sample comb...)
Microwave
UV transilluminator
Camera
Micro -pipet, Micro- pipet tip, Eppendorf tube

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2. Methods:
2.1: Restriction nucleases
- Prepare the interation mixture as follow:
Component
Smart mix
Deionized water
(it was prepared
10X Buffer
before)
TangoTM
BamHI, 10u/l
XhoI, 10u/l
DNA, 0.25 g/l
Final volume

Volume
3.0l
2.0l

6.0 l

0.5l
0.5l
4.0l
10.0l

- Incubate the reaction mixture at 37o C for 1 hour.

2.2: Preparing and runing agarose gel electrophoresis of DNA

To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration (0.8%), then heated in a microwave oven until completely melted.
After cooling the solution to about 60oC, it is poured into a casting tray containing
a sample comb and allowed to solidify at room temperature.
After the gel has solidified, the comb is removed. The gel, still in its plastic tray,
is inserted into the electrophoresis chamber and just covered with buffer.
Samples containg plasmid DNA mixed with loading buffer are then pipeted into
the sample wells.
Set up the electrophoresis apparatus and run at 50 ~ 100 V
The distance DNA has migrated in the gel can be judged by visually mornitoring
migration of the tracking dyes.
To visualize DNA, The gel containing DNA is stained by ethidium bromide, and
then the gel is observed on a UV transilluminator.

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C. RESULT AND DISCUSSION (GROUP 3.1)

Lane No.

Caption

Lane 1

DNA ladder (Marker)

Lane 2

Plasmid DNA

Lane 3

Plasmid DNA was treated with restriction enzyme BamHI

Lane 4
Lane 7

Plasmid DNA was treated with restriction enzyme BamHI and

XhoI (control samples)
USTHBI4-059

Nguyn Th Minh Hi

The rate of migration of DNA fragments in agarose is different:

Lane 1: The ladder DNA moves along from cathode (negative charge) to anode
(positive charge) all the length of the gel and on the band has many light trail.

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 Lane 2: There are two light trail in the gel and the distance between them is
close.
Lane 3: Only has one light trail.
Lane 4: There are two light trail in the gel and the distance between them is
further than lane 2.
Lane 7: is the same of lane 4.

The mechanism of migration and saparation:

The negative charge of phosphate backbond moves the DNA toward the positive
charge anode during electrophoresis. The gel matrix is responsible for the
separation of DNA by size during electrophoresis. The gel sieves the DNA by the
size of the DNA molecule whereby smaller molecules travel faster.
Comformation of DNA molecule also significantly affect the movement of the
DNA. For example, supercoiled DNA usually moves faster than relaxed DNA
because it is tightly coil and hence more compact.
In lane 2, which contain normal plasmid DNA preparation, multiple forms of DNA
is the reason why there are two light trail appeared. The gel from the
electrophoresis of the plasmid show a main band which would be the negative
supercoiled form, while orther forms of DNA appeared as a minor fainter bands.
There minor bands may be nicked DNA (open circular form) and the relaxed
closed circular form which normally run slower than supercoiled DNA, and the
linear strand form (in lane 3 which contain open circular form which was cut by
restriction enzyme BamHI in one site).

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In lane 4 and 7, which contain plasmid was cut into two fragments (all of them are
open circular and have different size, the bigger strand contains about 6.0 kb and
the smaller strand contains about 1.5 kb) by restriction enzyme BamHI and XhoI.
The movement of DNA in lane 4 and 7 follows: smaller molecule travel faster.

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D. CONCLUSION
Size of DNA being electrophoresis affects the migration of nucleic acid: smaller
molecules travel faster than larger molecules in gel.
DNA with different conformations that has not been cut with a restriction enzyme
will migrate with different speeds. Nicked or open circular DNA will move slowly
than linear and supercoiled DNA (slowest to fastest: nicked or open circular,
linear, super coiled plasmid).

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