Final Report of Molecular Biology

8/8/2019 Final Report of Molecular Biology

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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



2MASTERS LABORATORY TECHNIQUES 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 bydigestion 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 andimportant 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).

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Molecule

http://en.wikipedia.org/wiki/Molecule

http://en.wikipedia.org/wiki/Protein



3MASTERS LABORATORY TECHNIQUES 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 DNAsample 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



4MASTERS LABORATORY TECHNIQUES 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

g/

l- 1 - - - - -

γ DNA

5g/l2 - - - - -- 2

10 x Buffer2 2 2 2 2 2 2

Steile Water15 16 - - - - 15

EcoRI Enzyme

12g/l1 1 1 1 1 1 -

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



5MASTERS LABORATORY TECHNIQUES 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

B

4.9 18

< 10 kb

Supercoiled

4.4 19

2.67 23.5

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)

1- λ/ EcoR1

21.8 12

7.6 15

5.9 16.5

5.5 18

4.4 20

7- λ/Hind III

23.1 12

9.4 14

6.6 16

4.8 18



6MASTERS LABORATORY TECHNIQUES

Fig. 5 STANDARD RESULT

Fig. 4 RESULT GROUP 1



7MASTERS LABORATORY TECHNIQUES 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). Wedidn’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 Cloningeret al, 2008).



8MASTERS LABORATORY TECHNIQUES 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 9

th

(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 thestandard. Also DNA sequencing is another way to identity the plasmid DNAs by centre for

biotechnology information (NCBI).



9MASTERS LABORATORY TECHNIQUES 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]. [

Accessed on 19th

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.

http://www.springerlink.com/content/x842142662844n56/



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