Table of Content

Title Page No

1. ABSTRACT…………………………………………......3

2. INTRODUCTION………………………………………3

3. AIM ……………………………………………………......6

4. MATERIALS AND METHODS…………………………6

5. RESULTS………………………………………………6-8

6. DISCUSSION…………………………………………….9

7. REFERENCES............................................9

Recombinant DNA Technology

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 CORE MOLECULAR BIOLOGY

LABORATORY REPORT

Submitted by: Venkateswararao Mandava

Student Number: 09214762

Submited To: Prof.Tim Aldsworth

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Abstract:

Gene cloning has gained a lot of scope during the past few decades, as the technique is useful
in the production of various commercial and theoretical compounds. Gene cloning involves
introduction of manipulated foreign DNA or gene into cell and allowing it to replicate for
gene expression. The present experiment was carried to obtain clones of E.coli containing
recombinant DNA of amplicillin production containing foreign DNA. The technique mainly
involves isolation of plasmid vector( PUC 19) DNA from cells using lysozomes, restriction
of isolated DNA using restriction endonucleases (EcoRi), ligation of restricted DNA and
desired foreign DNA (λ genome) using DNA ligase. In this present experiment the EcoRI
was used to restrict the λ DNA and puc19 (vector). This fragment of DNA was
separated based on the size in agarose gel electrophoresis. The migration
distance of DNA was calculated and compared with the standard values. A graph
was plattered taking migration distance on x axis on logkb on y axis from
standard values which is useful for determining the size of PUC19. The λ DNA and
puc 19 were ligated using T4DNA ligase and transformed into culture DH5α using calcium
chloride and heat stock method.

Introduction:

Recombinant DNA technology was generated for the construction of transgenic

organisms.This technique is mainly applicable for the development of simulated dna.The
main grades associated with this technique are the cutting the dna, cleaving the restricted
strain and transcript the dna in to the bacterial cells. There are different methods to isolate the
Recombinant DNA from plasmid DNA. Agarose Gel Electrophoresis is the common method
used to separate the DNA molecule. The electrophoresis is defined as the movement of
charged molecules in the presence of electric field. In order to move the genetic information
from one organism to another living organism the following four steps were involved.

• Isolation

• Restriction

• Ligation

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 • Transformation

Isolation:

The process Isolation is defined as the separation of plasmid DNA from the host cells. The
circular plasmid DNA was transformed to liberalise by cutting with an enzyme called
restriction endonuclease. Plasmid is an extra chromosomal circular DNA fragment present in
prokaryotic cell such as bacteria and rarely in eukaryotic cell. Enzymes and detergents are
added to the suspension to dissolve cell walls of bacteria releasing both the bacterial DNA
and plasmid DNA molecules from the cells. The plasmid is also called as vectors which can
be ligate to DNA. The plasmid used in this experiment is PUC 19. The PUC 19 is a mutant
strain of Ecoli.
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Restriction:

Restriction comprises of breakdown of genetic material of DNA and RNA at particular size
known as recognition sites with the use of end nuclease enzymes. The enzymes serve as a
catalyst and cut the DNA molecule at phosphodiesterbonds. In the present experiment the
Cori was used as restriction enzyme which cut the circular DNA to linear single strands and
sticky ends.

Ligation:

Ligation is defined as conjugating the restricted fragments with the use of DNA ligase
enzymes. In this experiment T4 DNA ligase was used as an enzyme which was generated
from the T4 bacteriophage. The ligation reaction demands the three ingredients in addition to
water.

1. Two or more fragments either with blunt or sticky ends.

2. Buffer which contain ATP for production of energy.

3. T4 DNA ligase.

Transformation:

To amplify the gene of interest the recombinant DNA should reintroduce in living cells. This
transfer of genetic information by extra cellular pieces Of DNA in bacteria is called
transformation. Generally plasmid vector is able to replicate because it has its own replicate
origin (Griffith et.al. 2005). Common methods involved in transformation process are high
calcium concentration and heat shock. This replicated bacterial cell is then tested by growing
suitable medium under hygienic condition to find the presence of interested gene. In this
current experiment EcoRi strain DH5α is used for segregation of plasmid. Restriction is done
by the restriction enzyme called EcoR1. This restriction enzyme cuts the λ DNA on PUC 19
and ligation is done. The result from this experiment was analyzed by using agarose gel
electrophoresis and transformation technique.

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Aim:

To perform the cloning techniques for the development of recombinant DNA in the host cell
and to calculate the percentage of transformation and its efficiency.

Materials and Methods:

The materials and methods were followed according to the schedule as given in the core
molecular biology practical booklet 2010-2011.

Result:

The samples obtained from the carried experiments were loaded by gel plate and analyzed by
agarose gel electrophoresis and on measuring the distance travelled by λ DNA fragments the
size of the plasmid is observed.

Table1: The following table gives the information about components of mixtures placed in
the wells1-7.

Well-1 Well-2 Well-3 Well-4 Well-5 Well-6 Well-7

PUC19

λDNA+E.coR PUC19 E.cor1 DH5α+PUC19 DH5α without with ligase

i ligase

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Photograph of agarose gel:

Figure: 1 This photograph shows the distance migrated by DNA fragments in different wells
of agarose gel electrophoresis.

The well 1 was loaded with λDNA+E.coRi where no band was observed in this line. The well
2 was loaded with PUC19 where no clear band was observed. The well 3 was loaded with
PUC19 + EcoRi, no clear band was observed. The well 4 was added with DH5α +PUC19
where a small smear was observed from a distance of 11cm from the well. The well 5 was
loaded with DH5α where a small smear was observed at a distance of 10.5cm from the well.
Well 6 which is negative ligase show 2 bands at a distance of 6.0 and 6.9cm from the well.
Well 7 which is a positive ligase show 2 bands at a distance of 6.0 and 6.7 from the well.

Table: Number of colonies formed by transformants

S.no Plates No. Of white colonies No. Of Blue Colonies

Plate-1 (undiluted)

1 DH5α (negative 0 0
control)

2 Plate-2 (undiluted)

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 DH5α + pUC19

23 4

3 Plate-3 (diluted) 2D

DH5α + pUC19

2 1

4 Plate-4 (undiluted)

DH5α (with ligase)

24 4

5 Plate-5(undiluted) 4D

DH5α(with ligase)

0 1

Calculation:

Transformation efficiency = total number of cells growing on the petri plate

Amount of DNA spread on the petri plate (in µg)

For plate 4:- 28

0.5

=56 transforms/µg of plasmid.

Reference:

1. Brown, T.A. (2000). Essential molecular Biology (2nd ed., Vol.1). New York: Oxford
University Press.
2.Griffiths, A.J.F, Wessler S.R., Lewontin R.C., Gelbart W.M., Suzuki D.T & Miller,
J.H.2005. Introduction to Genetic Analysis (pp 341-379). (8th Ed). New York:
W.H.Freeman and company.

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3. Lodish et al. (2008). Molecular Cell Biology. (6th Ed). New York: W.H.Freeman and
Company.

4. Rapley, R & Walker, J. Core molecular Biology Practical Manual, (pp. 5-19) (2007-
2008). Hertfordshire: university of Hertfordshire.

5. Walker, J.M. (2002). SDS Polyacylamide Gel Electrophoresis of Proteins. The Protein
Protocols Handbook, 2, New Jersey: Humana press.

6. Wong Dominic, W.S. (2006). The ABCs of Gene Cloning. (2nd Ed). New York: Springer
Science+Business Media, Inc.