UNIVERSITY OF WOLVERHAMPTON

SCHOOL OF APPLIED SCIENCES

AGRICULTURAL BIOTECHNOLOGY

Report:
SMALL SCALE ISOLATION OF PLASMID DNA

Module Leader : P.Hooley

Module Name : MASTERS LABORATORY TECHNIQUES

MOLECULAR BIOLOGY (Report 1)
Module Code : AB4420
Student Name : JALAL OMER AHMED
Student Number : 0914607
Course Name : MSc Agricultural Biotechnology
2009-2010
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Abstract:
Isolate DNA from the plasmid is one of the common practices in research laboratories; this
procedure can be copied and cheap for the preparation of small-scale plasmid DNA. This is
based on alkaline lysis. Alkaline lysis of E.coli is the most common method for the isolationof
plasmid. This experiment is done in three steps, first step is alkaline lysis followed by
digestion with restriction enzyme lysozyme and finally separation based on sizes using
agarose gel electrophoresis. Through experiment we got the following results, two of these
four samples contain small plasmids (2.67 -4.9 kb in length < 10 kb), one contains the
fertility factor plasmid with approximately 94.5 kb. Also the remaining one is plasmid free.
The objective of this experiment is to identify these unknown samples of plasmid DNA.

Introduction:
E.coli is the sample organism that contains circular DNA molecules called plasmids.Plasmids
are usually much smaller in terms of the size of the chromosomes and vary in size from a
few thousand bp to several hundred thousand bp. Because of the small size of plasmids
compared to bacteria, it can be easily isolated and separated from the chromosomes by
using the special procedures, as alkaline lysis technique (Fig 1). This is one common
technique for plasmid purification, which breaks open with an alkaline solution proteins are
removed by precipitation and the plasmid DNA is recovered with alcohol precipitation.
Some plasmids analyzed the possibility of organic compounds and nitrogen fixation. Other
plasmids carrying antibiotic resistance genes and their spread in bacterial pathogens of
great medical importance. Plasmids used in molecular studies of different organisms and
important in many departments of biology, medicine, environment and development as
well as basic research in microbiology, and molecular biology.

The second step of the experiment is digestion with restriction enzymes. Restriction
enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and
then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes
play a very important role in the construction of recombinant DNA molecules. The most of
restriction enzymes recognise and cut a specific siteof double stranded DNA known as
restriction site, and also produce short stranded regions are known as sticky or cohesive
ends. For example EcoRI cuts between G and A bases at staggered position on each strand
and produce an overhangs of single stranded DNA are known sticky ends (Brown, T.A, 2006).
(Fig2)

The last stage of the experiment is electrophoresis which is a technique used in the
laboratory that results in the separation of (DNA), (RNA), or protein molecules. DNA is a
negatively charged molecule, and is moved by electric current through a matrix of agarose.
Agarose gel electrophoresis is a means of separating uncut or cut DNA molecules according
to their size. These fragments can then be isolated and purified from the gel matrix and
used for subsequent DNA cloning experiments. The gel was observed under the UV light
using Gel Doc 2000 system from BioRad (Khalil et al., 2003).
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Its migration in an electric field is proportional to its mass that is dependent relative to molecular
size, small DNA molecules can pass through the agaros gel easily than large molecules. In the
gel electrophoresis the supporting medium is used to uphold the nucleic acid sample. There are
some supporting matrices are available, but agarose is the largely used because of its easyness
to use and safe to prepare in the lab. The technique of electrophoresis is based on the fact that
DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when
an electrical potential is placed on the DNA it will move toward the positive pole. (Fig 3) the DNA
sample were mixed with gel loading dye 1X (Final concentration) and loaded into the wells.
The gel was run at a voltage of 1.5 V/cm for 1-2 hours and the bands were visualized under
UV light and photographed (Sambrook, 2001).

Fig.2 Restriction enzyme

Fig.3 Gel electrophoresis

Fig.1 Principle of Alkaline lysis

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Procedure (Changes):
a) In the fourth stage of the Protocol (Isolation of plasmid DNA), we used sodium
acetate instead of using the NaOAc.
b) In the seventh stage of the Protocol (Isolation of plasmid DNA), we should centrifuge
the tubes at 4 C but we did centrifuge at room temperature.
c) In the eighth stage of the Protocol (Isolation of plasmid DNA), we didn’t use the
vacum to dry the tubes; we used tissue to dry the tubes.
d) In the second stage of the Protocol (restriction and electrophoresis protocol),
according to the protocol after preparation the 7 tubes we have to keep the tubes in
water at 37 degree for 60 minutes but we kept the tubes for 45 minutes, due to run
out of time.
e) In the third phase of the Protocol (restriction and electrophoresis protocol),
according to the protocol it is more convenient to heat inactivate the digest by
incubating the microtubes at 65 degree for 5 minutes. However, we added 0.5M
EDTA PH7.5 to stop the reaction.

Table 1: Digestion Restriction and electrophoresis of DNA samples:

Tube No. 1 2 3 4 5 6 7

Students Sample - - 17(A) 17(B) 17(C) 17(D) -

pBR322
- 1 - - - - -
1g/l
γ DNA
2 - - - - -- 2
5g/l
10 x Buffer
2 2 2 2 2 2 2
Steile Water
15 16 - - - - 15
EcoRI Enzyme
1 1 1 1 1 1 -
12g/l
HindIII Enzyme - - - - - - 1

Total 20 l 20 l 20 l 20 l 20 l 20 l 20 l

Also used four standard solutions were prepared in advance:

 Tube 8, 5 l LambdaR1/HindIII
 Tube 9, 5 l LambdaR1/Uncut
 Tube 10, 5 l LambdaR1/EcoR1
 Tube 11, 5 l pBR322/Uncut
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Result:
The given plasmids are of 3 different conformations like supercoiled, open circular and
linear. Due to the enzymatic and shear induced breakup of backbone sugar phosphate
chain, the open circular and linear molecules have been resulted. This operation ran on Gel
electrophoresis and measured the distance of the migrant plasmids (Kaichun Zhua, et.al.
2005). Plasmid size is estimated by plotting a graph between distances travelled but the
plasmid and fragment size.

From our gel result, we didn’t get result of unknown samples (Fig. 4). From the standard gel,
the two wells 7 and 8 (B, C) <10kb, the plasmid sizes are 4.9, 4.4, 2.67 kb, 2.65 kb
respectively. The well 6th (A) is plasmid free as no bands present. Well 9th (D), is fertility
factor with size approx... 94 kb. (Graph 1)

Table 2: Estimation of the sizes of isolated plasmids (Unknown Standard A, B, C and D)

Tubes Molecular Size(Kb) Distance (mm) Comments

A Plasmid free - Plasmid free
4.9 18
B 4.4 19 < 10 kb
2.67 23.5 Supercoiled
C 2.85 23
D >94.5 11.5 Fertility factor(linear)
By plotting the graph between distances travelled to the fragment sizes. Among 11
ependorf tubes, 4 tubes are standards and 7 are our samples. As depicted in the in the
table1.

Table 3: Estimation of the sizes of isolated plasmids (λ/ EcoR1, λ/Hind III)- (Our results)

Tubes/ enzymes Molecular Size(Kb) Distance (mm)

21.8 12
7.6 15
1- λ/ EcoR1 5.9 16.5
5.5 18
4.4 20
23.1 12
9.4 14
7- λ/Hind III
6.6 16
4.8 18
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Fig. 4 RESULT GROUP 1

Fig. 5 STANDARD RESULT

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Discussion:
The given plasmids are of 3 different conformations like supercoiled, open circular and
linear. Due to the enzymatic and shear induced breakup of backbone sugar phosphate
chain, the open circular and linear molecules have been resulted. This operation ran on Gel
electrophoresis and measured the distance of the migrant plasmids (Zhua, et.al. 2005). We
didn’t get result for our samples A, B, C and D because of the following unexpected reasons
as per my opinion. The reasons could be:

 Given plasmid samples might be contaminated.

 Apparatus used like pippettes; eppendorf tubes... might be contaminated.
 The chromosomal DNA, Bacterial debris and proteins might not be efficiently
removed during centrifugation.
 Some particles like proteins, bacterial debris might be remained in lysate after
centrifugation.
 Might be not efficiently removed alcohol by the tissue.
 Might be due to changes in the protocol.
 Probably due to technocal error.

1. The reason behind the usage of glucose is to provide growth media to the plasmids and
to maintain osmolarity of the system and to prevent the cell from bursting immediately.
The role of lysozyme is to break down the Bacterial cell wall and to initiate plasmid DNA
isolation process (Old and Primrose, 2001). Polymeric compounds offers rigidity to the
cell wall, so lysozymes digests these polymeric compounds and weakens cell membrane
(Brown, T.A, 2006).
GTE buffer solution is a mixture of glucose, Tris and EDTA.

Glucose- maintains osmolarity of the cell

Tris- buffering agent i.e. maintains pH (8.0)

EDTA- prevents degradation by binding to divalent cations which are necessary for
DNAses activity (Primrose. S. B. et.al. 1994).

2. NaOH offers a narrow range of alkalinity to the solution i.e. 12.0-12.5, at which non
supercoiled DNA denatures but supercoiled plasmid DNA has no effect. Upon NaOH
addition, the hydrogen bonds in non supercoiled DNA molecules are broken as a result
of unwinding of double helix. NaOH concentration is necessary to ensure complete
conversion of chromosomal DNA to single strands. Here the pH of lysis solution is 12.5.
Hence it’s mandatory that plasmids must be released into NaAc which neutralises
partially and reduce the pH below threshold pH i. e 12.3. NaOH removes lipid molecules
and helps in easier disruption of cell wall. Covalently closed plasmid molecules remains
unaffected upon NaOH addition, by centrifugation the unwanted chromosomal DNA,
protein content and bacterial debris is easily separated (Brown, T.A, 2006 and Cloninger
et al, 2008).
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3. Gel electrophoresis work by making use of electric charges to distinguish molecules.

When DNA placed in an electric field, they run towards positive charge as they being
negatively charged. The migration rate of plasmids depends upon sizes i.e. smaller ones
travel more distance than bigger. We were provided 3 conformations such as
supercoiled, circular and linear. During plasmid replication, supercoiling occurs by
enzyme topoisomerases. But circular molecule (CCC, OC) runs faster than any other.
Moving through pores matrix is depending on size as smaller moves faster. Covalently
closed molecules run faster than open circular plasmids. Linear molecules (L) like
fragmented chromosomal DNA have no such topological constraints. Linear molecules
move zig-zag like snaking action through Agarose gel. This is the reason why circular ones
travel faster than others. But in EcoR1, linear forms run faster just ahead of open circular
forms as EcoR1 has only a single target site. (Old and Primrose, 2001).
As shown from fig 4 in our gel result, we didn’t get the result for 4 unknown samples.
However, as shown in (graph 1) from the standard gel, the well 6th (A) is plasmid free as
no bands present. The two wells 7 and 8 (B1, B2, B3 and C) <10kb, the plasmid sizes are
4.9, 4.4, 2.67 kb, 2.85 kb respectively. Well 9th (D), is fertility factor with size approx...

4. Plasmid DNA content is very less compared to bacterial DNA. Plasmid amplification offers
an increasing yield of DNA i.e. plasmid copy number. Plasmids can be able to replicate in
absence of protein synthesis of host bacterium. But bacterial chromosome cannot
replicate like this upon the addition of inhibitors like chloremphenicol. On the addition of
inhibitors, bacterial chromosomal replication and cell division are blocked, yet plasmid
molecules continue to replicate. Hence, in presence of inhibitors, we can able to get
considerable multicopies of the plasmid DNA. This is the sophisticated method of getting
large number of plasmid copies for plasmid DNA isolation. (Brown, T.A, 2006).

5. The steps to identify the plasmids are as follows :

a) Separation on the basis of size
b) Digestion with Restriction enzymes
c) Running Gel electrophoresis.

We can use restriction enzymes like HindIII, EcoR1 etc.. These restriction enzymes allow
us to verify the plasmid based on size followed by Gel electrophoresis and southern
blotting etc.. By Gel electrophoresis, we can estimate plasmid size according to the
standard. Also DNA sequencing is another way to identity the plasmid DNAs by centre for
biotechnology information (NCBI).
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References:

1. Brown, T. A (2006) Gene cloning and DNA analysis 5th Ed. Blackwell Publications.
Oxford. P p 39-47.

2. Cloninger. C, Felton. M, Paul. B, Hirakawa. Y, Metzenberg. S. (2008). Control of pH

during plasmid preparation by alkaline lysis of Escherichia coli. Analytical
Biochemistry. 378. P p224-225.

3. Khalil .A.B, Anfoka .G.H., and Bdour.S (2003) Isolation of plasmids present in
thermophilic strains from hot springs in Jordan. World Journal of Microbiology &
Biotechnology. [online].19 (1) pp.239-241 [Accessed on 21st February 2010].
Available at < http://www.springerlink.com/content/x842142662844n56/ >.

4. Lyses of bacterial cells for plasmid purification. QIAGEN in United Kingdom [online]. [
th
Accessed on 19 February 2010 ] Available at :<
http://www1.qiagen.com/literature/qiagennews/0299/992lysis.pdf >.

5. Old and Primrose (1994) Principles of gene manipulation 5th Ed.Blackwell Scientific
Publications, Oxford and Northampton. P p 46-52.

6. Sambrook . J, Russel. D.W (2001) Molecular cloning: a laboratory manual.” Cold

Spring Harbor Laboratory Press, pp. 8.13-8.16.

7. Zhu. K, Jin. H, Ma. Y, Ren. Z, Xiao. C, He. Z, Zhang. F, Zhu. Q and Wang. B. (2005). A
continuous thermal lysis procedure for the large-scale preparation of plasmid DNA.
Journal of Biotechnology. 118. P p257–264.