CONTENTS

1. Abstract
2. Introduction
3. Plan of work
4. Materials and Methods

i) Bacterial Genomic DNA

ii) Plasmid Isolation

iii) Polymerise Chain Reaction

iv) Restriction Enzyme Digestion

v) Ligation

vi) Competent cell preparation and transformation

vii) Transformation expression in host cell

viii) IPTG Induction

ix) SDS PAGE

Results and Discussion

Conclusion

References

Chapter 1: Abstract

The general objective of cloning is to express specific proteins in vitro to study genes and gene
manipulation effects. The process starts with cloning a gene of interest (GOI) into a plasmid that
contains the necessary elements for propagating the plasmid/GOI in bacteria, and later
expressing the GOI in either prokaryotic or eukaryotic cells.

The plasmid used was pET21b in XL Blue strain of Escherichia coli bacteria. We amplified the
isolated plasmid using PCR. The amplified plasmid was cut using restriction enzymes and
ligated to obtain the required gene, viz. eGFP. This gene was incorporated into competent cells.
The competent cells were then induced to produce the protein (eGFP), with the help of IPTG
(Isopropyl β-D-1-thiogalactopyranoside). The protein was then analysed through PAGE.

Chapter 2: Introduction

Gene cloning is a set of experimental methods in molecular biology that are used to assemble
recombinant DNA molecules and to direct their replication within host organisms. The use of the
word cloning refers to the fact that the method involves replication of a single DNA molecule
starting from a single living cell to generate a large population of cells containing identical DNA
molecules. Steps involved in cloning includes choice of host organism and cloning vector,
preparation of vector DNA, preparation of DNA to be cloned, creation of recombinant DNA with
ligase, introduction of recombinant DNA into host organism, selection of organism containing
vector sequence, screening for clones with desired DNA inserts and biological properties. It can
be used for production of recombinant proteins, genome organisation and gene expression.

Prokaryotes are a group of organisms whose cells lack a membrane-bound nucleus (karyon).
Those organisms whose cells have a well-defined membrane-bound nucleus and organelles are
called eukaryotes. Most prokaryotes are unicellular organisms, although a few such as
myxobacteria have multicellular stages in their life cycles or create large colonies like
cyanobacteria. Prokaryotes do not have a membrane-bound nucleus, mitochondria, or any other
membrane-bound organelles. In other words, all their intracellular water-soluble components
(proteins, DNA and metabolites) are located together in the same volume enclosed by the cell
membrane, rather than in separate cellular compartments.

Although a very large number of host organisms and molecular cloning vectors are in use, the
great majority of molecular cloning experiments begin with a laboratory strain of the bacterium
Escherichia coli (E. coli) and a plasmid cloning vector. E. coli and plasmid vectors are in
common use because they are technically sophisticated, versatile, widely available, and offer
rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is
exceptionally large (hundreds of thousands to millions of base pairs) then a bacterial artificial
chromosome or yeast artificial chromosome vector is often chosen.

Specialised applications may call for specialised host-vector systems. For example, if
experimentalists wish to harvest a particular protein from the recombinant organism, then an
expression vector is chosen that contains appropriate signals for transcription and translation in
the desired host organism. Alternatively, if replication of the DNA in different species is desired
(for example transfer of DNA from bacteria to plants), then a multiple host range vector (also
termed shuttle vector) may be selected. In practice, however, specialised molecular cloning
experiments usually begin with cloning into a bacterial plasmid, followed by sub-cloning into a
specialised vector.

STEPS IN GENE CLONING:

1) Firstly, we need to acquire a sufficient quantity of the DNA desired to be cloned. Well, if we
had a little bit of the DNA and knew enough of the sequence to make primers, we could use PCR
(Polymerization Chain Reaction) to amplify it.

If we started with RNA (e.g. mRNA) then we would need to use reverse transcriptase to 'reverse
transcribe' it into DNA then PCR amplify it.

2) Next we decide on a vector. A vector is usually a piece of DNA that replicates in a bacterium.
Plasmids are usually used, they are small pieces of circular DNA that are often found in bacteria
which are self-replicating and are maintained in the cell in a stable and characteristic number of
copies. Some bacteria have very high numbers of plasmids which make them ideal cloning
vectors. So, you can see, a gene inserted into a plasmid will be replicated many of times in a
single bacterium. And it is possible to grow millions of bacteria in a 1 liter flask. So this is a
good way to make lots of copies of a gene of interest. Routinely in the lab a weakened strain of
the bacteria E. coli is used for these experiments. If it is not grown in the lab under special
conditions it will quickly die.

3) Application of restriction digestion to cut the DNA: Next we use restriction enzymes to cut
out the gene we want from the amplified product. We use the same enzymes to cut the vector so
that the ends will be compatible. This means that if we use a "sticky ends" restriction enzyme,
then the DNA sequences of the trailing bits will be compatible and will want to stick to each
other. This makes it much easier to do. If we can't find a suitable site for a sticky end restriction
enzyme then we can use a "blunt-ended" one, which is a bit harder to make work. Once the
restriction is done the piece of DNA that you want to clone needs to be separated from the parts
you do not want this is done using gel electrophoresis as this will make it easier to clone. This is
usually done by running the digest out on a gel and physically cutting out the fragment that you
want with a scalpel.

4) Ligation: Once both the vector and the target DNA have been cut we mix them together and
add the ligase enzyme. This enzyme ligates (connects) the phosphodiester backbone acting as a
glue to stick the ends together.

5) Transformation: This just means add the DNA back into a bacterium. There are a number of
different ways of doing this, but most of them involve making the bacterial cell membrane
temporarily porous so that it can take up the liquid containing the DNA mix. Then you let the
cells recover and grow them up as usual.

6) Analysis: Sometimes we don't get the expected expression. For instance, a sometimes time the
vector will just stick back to itself, or stick to another vector without having any target DNA in
it. So you have to have a mechanism for picking out the ones where the target DNA has gone
into the vector.
Chapter 3: Plan of work

Chapter 4: Materials And Methods

1. Bacterial Genomic DNA Isolation

The isolation and purification of gDNA from the cells is one of the most common procedures in
molecular biology and embodies a transition from cell biology to the molecular biology from in
vivo to in vitro , as it were in genomic DNA isolation the need is only to separate the total DNA
from RNA, protein, lipid, etc.

The CTAB extraction procedure includes Lysis of the cell membrane, Extraction of the genomic
DNA and Precipitation.

Chemicals and Reagents:

 CTAB
 β-mercaptoethanol
 EDTA
 Tris-HCl
 Absolute alcohol
 Chloroform
 Iso-amyl alcohol
 TE buffer 10X (assay buffer)
 Gel loading buffer 6X
 Sterile distilled water
 Agarose powder
 1X TAE buffer
 DNA ladder 1kb
 Ethidium bromide stock (1mg/ml)
Instruments:

 Centrifuge
 Electrophoresis unit
 Powerpack
 Weighing balance
 Microwave
 UV-Transilluminator
 Water bath (37°C)

Miscellaneous

 2ml Eppendorf
 Eppendorf rack
 Crushed ice
 Micropipettes and sterile micro tips
 Gloves

Role of each reagent:

Tris: Interacts with the lipopolysaccharides present on the outer membrane which helps to
permeabilise and also acts as a buffering reagent.

EDTA: It chelates the divalent cations of DNases and hence keeps the DNA in the solution, it
binds to Ca2+ and prevents joining of cadherins between cells, preventing clumping of cells
grow in liquid suspension.

Absolute ethanol: Removes the water of hydration. So the DNA cannot dissolve and sets free in
the solution. And ethanol wash helps remove salts and any remaining SDS as these can interfere
with a restriction digestion.

Chloroform: Isoamyl alcohol (24:1): removes protein by precipitating it nut leave DNA in
aqueous solution.

TE buffer: 10mM Tris-Cl (desiring pH) 1mM EDTA (pH8.0) Tris buffers the DNA solution.
EDTA binds divalent cations (especially Mg+2 ions) that are a needed as cofactor for bacterial
nucleases and thus limits DNA degradation.

Buffer preparations:
 1. CTAB buffer composition:

Sr.No.

Components

Volumes

2% CTAB (hexadecyltrimethylammonium bromide)

20 ml from 10% CTAB buffer

100 mM Tris-HCl (pH 8.0)

10 ml from 1M Tris-Cl (pH 8.0)

20 mM EDTA (pH 8.0)

4ml from 0.5M EDTA (pH 8.0)

1.7 M NaCl

34ml from 5M NaCl

0.3% β-mercaptoethanol (add just before use)

300µl from liquid β-ME

Total

100ml

1. 10% CTAB
2. 10g of CTAB
3. Add 75ml of D/W
4. Heat the solution at 60°C for 15mins
5. Stir till CTAB dissolves completely.
6. Bring total volume to 100ml with D/W
 1. TE buffer
2. 1ml 1 M Tris HCl Ph 8.0
3. 0.2ml 0.5M EDTA Ph 8.0
4. Bring total volume to 100ml with D/W.

1. 1 M Tris HCl Ph 8.0

2. 121.1 g Tris
3. Dissolve in about 700ml of D/W.
4. Bring pH down to 8.0 by adding 10N HCl (about 50ml).
5. Bring total volume to 1 L D/W.

1. 0.5 M EDTA pH 8.0

2. 186.12 g EDTA
3. Add about 700ml of D/W
4. Adjust the pH to 8.0 by 10 N NaOH
5. EDTA won't dissolve until the pH is near 8.0
6. Bring total volume to 1 L with D/W.

1. 5 M NaCl:
2. 292.2 g NaCl
3. 700 ml D/W
4. Dissolve and bring to 1 L.

Method:

1. Take 1ml of bacterial culture in 2ml Eppendorf.

2. Centrifuge at 20000 rpm for 5mins.
3. Discard supernatant and collect pellet and add 1ml CTAB
4. Keep the Eppendorf at 60°C for 30minutes.
5. Centrifuge the tube at 10000rpm for 5 minutes
6. Collect the supernatant into two tubes (1ml in each tube).
7. Add 1ml of Chloroform: Isoamyl alcohol (24:1) and mix slowly.
8. Centrifuge at 10000rpm for 5minputes.
9. Collect upper aqueous layer into 2ml Eppendorf tube & again add 1ml of Chloroform:
Isoamyl alcohol (24:1), mix slowly.
10. Add 2 volumes chilled absolute alcohol.
11. Collect upper aqueous layer into new 2ml Eppendorf tube
12. Add 2 volumes chilled absolute alcohol.
13. gDNA will get precipitated, collect DNA in a new tube with the help of glass rod or
micropipette.
14. Add 500µl of chilled absolute alcohol.
15. Centrifuge for 5minutes at 10000rpm
 16. Discard supernatant and dry the pellet at 37°C
17. Reconstitute with 150µl of TE buffer.
18. Load 10µl on AGE.

1. PLASMID ISOLATION

 Plasmid is a double-stranded, circular extrachromosomal DNA of bacterium. It is used in

recombinant DNA experiments to clone genes from other organisms and make large
quantities of their DNA.
 Size of plasmids ranges from 1-1000 kilobase pairs.
 The term 'plasmid' was introduced by American molecular biologist Joshua Lederberg.
 They are considered as transferable genetic elements or 'replicons'. They are actually
naked DNA.
 Mainly there are two types of plasmids: conjugative and non-conjugative. Conjugative
genes have trans-genes (trans-transfer) and can perform conjugation. Non-conjugative
plasmids cannot perform conjugation. There is an intermediate class of plasmids called
mobilisable plasmid. Mobilisable plasmids can carry only a subset of genes required for
transfer.
 The cells are lysed using an alkaline lysis procedure. The cells are brought to a high pH
to not only lysed the cells, but also to denature the DNA. The DNA solution is then
neutralised. Since plasmid DNA is circular and supercoiled, when the pH is brought back
to neutral, the plasmid DNA snaps back to being double-stranded. By contrast, genomic
DNA is so large that it is broken into linear pieces.

AGAROSE GEL ELECTROPHORESIS:

In order to visualise the extracted plasmid, its quality as well as its quantity, we run it on agarose
gel. Agarose is a polysaccharide derived from seaweed that will dissolve in liquid when boiled
and then solidify to form a gel when it cools. DNA fragments will move through such a gel when
exposed to an electric current, and the rate at which they move depends on their size: larger the
fragment, the slower it moves. Most agarose gels used a buffer called TAE (Tris/acetate/EDTA).
Tris is a commonly used buffering agent since it is biologically inert. In this case it is buffered
with acetic acid, though other acids would also work. EDTA is a compound that binds divalent
metals, such as Mg2+. Since most nucleases capable of attacking DNA need Mg++ this
effectively inactivates them.

1. Nucleic acids are not chemically altered during the size separation process.
2. Can easily be viewed and handled.
3. Samples can be recovered and extracted from the gels easily for further studies.
4. Gel could be stored in a plastic bag and refrigerated after the experiment.

It also has a limitation that a lot of heat is generated during the run.

Materials:
For plasmid isolation:

1. Overnight grown culture of XL Blue containing pET21b plasmid at an OD620 of 1.00-

1.20.
2. Sterile Eppendorf tubes.
3. Solution 1.
4. Solution 2A and 2B.
5. Solution 3.
6. Chloroform.
7. Isoamyl alcohol.
8. Isopropanol.
9. 70% ethanol.
10. Absolute alcohol.

For agarose gel electrophoresis

1. Agarose (for 1% gel).

2. Ethidium bromide (1mg/ml)
3. RNase (100μg/ml).
4. 1X Tris-acetate EDTA buffer.
5. Power packs.
6. Electrophoretic apparatus.
7. Benchtop centrifuge.
8. Micropipettes.
9. Micro tips.
10. Dry bath at 37°C.
11. Dry bath at 55°C.
12. Weighing balance.
13. Spatula.
14. Weighing boat.
15. 10X assay buffer.
16. Gel loading buffer (6X)
17. St. distilled water.
18. DNA ladder 1kb
19. Microwave.
20. Gloves.
21. Ice or frosty box.

PROTOCOL:

PLASMID ISOLATION

1. Take 1ml culture in 1.5 ml Eppendorf tubes. Spin at 10000 rpm for 7minutes.
2. Discard the supernatant and remove the remaining media using 50µl pipette.
3. To this add 100µl of solution 1 and mix.
4. After complete resuspension, add 200µl of solution 2 (equal volumes of 2A and 2B)
Mix gently.

1. To this add 150µl of solution 3. Mix gently for 1 minute. Spin at 10000 rpm for 7mins.
2. Carefully aspirate the supernatant and note the volume. To it, add equal volume of chilled
Chloroform: Isoamyl alcohol (24:1).
3. Centrifuge at 10,000 rpm for 7 minutes.
4. Carefully aspirate the supernatant into another tube and note the volume. To it, add 0.6
volume of chilled isopropyl alcohol.
5. Centrifuge at 10,000 rpm for 10 minutes. Discard the supernatant.
6. To the pellet add 300µl of chilled 70% ethanol. Carefully mix without shearing the pellet.
7. Centrifuge at 10000 rpm for 7 minutes.
8. Decant the supernatant and add 300µl of absolute alcohol. Carefully mix without
shearing the pellet.
9. Centrifuge at 10000 rpm for 7 minutes.
10. Decant supernatant and dry the pellet in dry bath at 37°C.
11. Reconstitute the pellet in 30µl of TE buffer.
12. Set 1: Load 10μl sample onto 1% agarose gel using 5μl of 1mg/ ethidium bromide.
13. Set 2: Do RNase Treatment.

PROTOCOL: RNase TREATMENT

1. To the 30µl of reconstituted sample, add 5µl of 1mg/ml RNase A.

2. Keep it in dry bath at 55°C for 45mins.
3. Remove sample and load 10µl sample onto 1% agarose gel using 5µl of 1 mg/ml
ethidium bromide.

1. POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a
single piece of DNA, generating thousands to millions of copies of a particular DNA sequence
and was developed by Kary Mullis in 1983. It exploits the natural function of polymerase present
in all living organisms to copy genetic material or perform 'Molecular Photocopying'.

BASIC COMPONENTS OF PCR REACTION MIXTURE INCLUDE:

1. Template DNA
2. Oligonucleotide primers
3. PCR reaction buffer
4. dNTPs (deoxynucleoside triphosphates)
5. Taq polymerase
6. PCR instrumentation and thermal cycler (Make: Applied Biosystems)

STEPS INVOLVED IN PCR:

 1. Initial Denaturation: Complete Denaturation of template DNA at the start of the PCR is
of key importance. Incomplete Denaturation results in inefficient utilisation of template
in the first amplification cycle and in a poor yield of PCR products. It is generally
performed at 94°C for 1-3min, depending on the G-C content of the template. If longer
initial Denaturation or temperature is necessary then Taq polymerase can be added after
this step as higher temperature may affect the enzyme stability. Initial Denaturation step
is performed only at the beginning of the reaction.
2. Denaturation: Subsequent Denaturation steps are performed for a shorter time of 30secs-
1min at 94°C for 30 cycles. This is sufficient since the PCR product synthesised in the
first amplification cycle is significantly shorter than the template DNA and completely
denatures under these conditions. Certain additives like DMSO or glycerol or formamide
are used to facilitate DNA Denaturation depending on the G-C content.
3. Annealing: The optimal annealing temperature is generally 5°C lower than the melting
temperature of primer-template DNA duplex, performed for 30secs-1min. If non-specific
products are obtained in addition the expected product; the annealing temperature is
optimised by increasing it step-wise 1-2°C.
4. Extension: Primer extension, resulting in synthesis of new DNA strand is carried out
72°C, which is optimal temperature for Taq polymerase activity. The amplification time
is determined by the length of the sequence to be amplified. For every 1kb target DNA,
1min time is recommended. Number of PCR cycles depends on the amount of template
DN in the reaction mixture and on the expected yield of the product.
5. Final Extension: As DNA synthesis proceeds, it becomes less efficient as most of the
components get used up. Hence, following the last cycle, enzyme is allowed to finish any
incomplete synthesis by carrying out a final extension at 72°C for 5-15min.
6. Final hold: This step 4-15°C for an infinite time may be employed for short-term storage
of the reaction.

MATERIALS REQUIRED:

1. Chemicals and reagents:

2. Taq DNA polymerase buffer
3. dNTPs (dATP, dGTP, dCTP, dTTP)
4. Forward primer, Reverse primer
5. Taq DNA polymerase
6. Ethidium bromide
7. Agarose
8. Gel loading buffer
9. DNA ladder

1. Instruments:
2. Thermocycler (Make: Applied Biosystems)
3. Electrophoresis unit
4. UV transilluminator
5. Microwave
6. Weighing balance
 7. Powerpack

1. Miscellaneous:
2. Gloves
3. Micropipettes and their tips
4. PCR tubes
5. Cello tape

PROTOCOL:

1. Preparation of reaction mixture for PCR:

2. Add reagents given in the kit as shown in the order in the table.

Reagents

Volume for 25μl reaction mixture

Distilled water

16.5

10X Taq polymerase assay buffer

2.5

25mM MgCl2

2.0

10mM dNTPs

0.5

Forward primer (10μM)

1.0

Reverse primer (10μM)

1.0

Taq DNA polymerase(5U/μl)

0.5

DNA template
1.0

Total reaction mixture volume

25.0

1. Mix the contents gently and tap spin the master mixture. Place the PCR tubes in the
thermal cycler machine for PCR amplification.

Steps

Temperature

Time(min)

Initial denaturation

95°C

Denaturation

94°C

Annealing

68°C

Extension

72°C

Final extension

72°C

Hold

4°C

∞
 1. Carry out the amplification using the following conditions and perform at least 33 cycles
for amplification of the NA fragment.
2. Load the samples on 1.5% agarose gel.

1. RESTRICTION ENZYME DIGESTION

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts the double-stranded or
single-stranded DNA at specific recognition nucleotide sequences known as restriction sites.

To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate
backbone (i.e. each strand) of the DNA double helix. Such enzymes, found in bacteria and
archaea, provide a defense mechanism against invading viruses. Inside a bacterial host, the
restriction enzymes selectively cut up foreign DNA in a process called restriction; the host DNA
is methylated by a modification enzyme (methylase) to protect it from the restriction enzyme's
activity. Collectively, these two processes form the restriction-modification system.

This self-protection is achieved by the help of the specific DNA methyltransferase enzyme
which will methylate the specific DNA sequence for its respective restriction enzyme
transferring methyl groups to adenine or cytosine residues to produce N-6-methyladenine or 5-
methylcytosine. Another interesting feature of restriction endonucleases is that they commonly
recognise recognition sequences that are mostly palindromes- they show the same forward (5; to
3' on the top strand) and backward (5' to 3' on the bottom strand) sequences. In other words, they
are nucleotide sequences or complementary strands that read the same in opposite directions.

FACTORS AFFECTING RESTRICTION ENZYME ACTIVITY:

1. Temperature: most digestions are carried out at 37°C. However, there are a few
exceptions e.g. digestion with Sma I is carried out at lower temperatures (~25°C), while
Taq I at higher temperature i.e. 65°C.
2. Buffer systems: Tris-HCl is the most commonly used buffering agent in incubation
mixtures, which is temperature-dependent. Most restriction enzymes are active in the pH
range 7.0-8.0.
3. Ionic conditions: Mg2+ is an absolute requirement for all restriction endonucleases, but
the requirement of other ions (Na+/K+) varies with different enzymes.
4. Methylation of DNA: Methylation of specific adenine or cytosine residues within the
recognition sequence of the restriction enzyme affects the digestion of DNA.

MATERIALS REQUIRED:

1. Restriction enzymes : EcoRI and EcoRV

2. Ice or frosty box
3. PCR vials
4. Labels
5. St. distilled water
 6. 10X assay buffer
7. DNA sample
8. Micropipettes and their tips

PROTOCOL: Restriction Enzyme Digestion:

1. Arrange all the reagents on the icebox.

2. Arrange the PCR vials in the rack and label them accordingly.
3. Add all the reagents into the PCR vials according to the reaction mixture provided below.

Chemicals

Volume (in μl)

Sterile distilled water

10X assay buffer

DNA sample

10

Restriction enzyme (EcoRI)

0.5

Restriction enzyme (EcoRV)

0.5

Total volume

25

1. Mix the contents by tapping or gentle vortexing and incubate the tubes at 37°C for 30
minutes in heat.
2. Load the sample on 1% agarose gel containing 5µl of ethidium bromide [1mg/ml]

PROTOCOL FOR AGAROSE GEL ELECTROPHORESIS:

1. Weigh 0.5g agarose and dissolve it in 50ml TAE buffer.

 2. Digest it in microwave till the agarose completely dissolves
3. Cool the gel to about 60°C
4. Fix the tray in the electrophoretic apparatus and make sure there is no leakage
5. Place the comb in the tank
6. Add 5µl of ethidium bromide and mix well
7. Pour the gel into the tray and allow it to solidify
8. Carefully remove the comb without disturbing the wells
9. Loosen the screw from the electrophoretic apparatus and place the tray into the tank
containing 1X TAE buffer
10. Load the samples into the well along with an appropriate marker
11. Connect the electrode
12. Plug the electrodes to the power pack and switch on the electric supply at a constant
voltage of 100V
13. Allow the dye to run up to ¾ of the gel
14. Switch of the power supply, carefully remove the tray from the tank and place the gel in
the gel doc unit
15. Switch on the UV lamp to analyse the gel.

1. Ligation:

DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of
making new viable DNA or plasmids. There are currently 3 methods for joining DNA fragments
in vitro. The first of these is DNA ligase that covalently joins the annealed cohesive ends
produced by certain restriction enzymes. The second depends on the ability of DNA ligase from
phage T4-infected E.coli to catalyse the formation of phosphodiester bonds between sticky or
blunt end fragments. The third utilises the enzyme terminal deoxynucleotidyl transferase to
synthesise homopolymeric 3' single-stranded tails at the ends of fragments. The most commonly
used is the T4 DNA ligase method.

Materials required

 Vials and vial stand

 Micropipette
 Pipette tips
 Vector
 Insert
 10X ligase buffer(µl)
 T4 DNA ligase (5 Weiss U/ml) (µl)

Protocol

Size of the vector: 5400 bp

Size of the insert: 750 bp

Amount of vector DNA to be taken for ligation= 200 ng

Concentration of the insert= 250 ng

Amount of insert to be taken for ligation= size of insert × amount of vector DNA

Size of vector

= 27.7 ng /µl

Concentration of insert for 1:3= 0.4 µl

1:5= 0.65 µl

1:10= 1.3 µl

Reaction volume: 10 µl

1:1

1:3

1:5

1:10

Ligation control

Vector volume (µl)

Insert volume (µl)

0.277

0.831

1.38
2.77

D/W (µL)

5.723

5.17

4.62

3.23

1OX ligase buffer (µl)

T4 DNA ligase (5 Weiss U/ml) (µl)

Total volume (µl)

10

10

10

10
10

Incubate the vials at room temperature for two hours.

Load the samples on 1% agarose gel and observe for ligation.

1. Transformation:

Transformation is a process in which the cells take up foreign DNA. Since DNA is a very
hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order for
this to occur, cells need to be competent. To be competent means that DNA can easily pass
through cell walls, thus readily incorporating foreign DNA material. Once the foreign DNA is
incorporated into the cell, it can be replicated along with the host's DNA if there is an origin of
replication or an antibiotic resistance gene.

There are two major parameters involved in efficiently transforming a bacterial organism. The
first is the method used to induce competence for transformation. The second major parameter is
the genetic constitution of the host strain of the organism being transformed. The ability to take
up DNA or competency must be induced by chemical methods using divalent and multivalent
cations (calcium, magnesium, manganese, rubidium, or hexamine cobalt)

1. Transformation in Host cell

Materials

 E.coli cells (TS XL BLUE)

 St. Luria Bertini broth
 Pre chilled sterile Eppendorf tubes
 Cold calcium chloride (0.1 M)
 Cold magnesium chloride (0.1 M)
 Ice bucket
 Cooling microcentrifuge
 Microcentrifuge tubes
 Micropipette
 Micro tips

Protocol

1. Inoculate 500 µl of overnight grown culture into 100 ml LB broth and grow at 37°C, 200
rpm.
2. Monitor the growth until it reaches 0.45-0.47 O.D600 using sterile LB broth as blank.
3. Quickly immerse the flask in ice and swirl.
 4. Add 2ml of culture in prechilled 2ml Eppendorf tubes.
5. Spin the cells at 5000 rpm/ 4°C/ 10 mins. Discard the supernatant.
6. Resuspend the cell in 300 µl st. cold 0.1 M MgCl2, till pellet completely dissolves.
(Flicking the tubes slowly).
7. Centrifuge the cells at 2500 rpm/4°C/ 10mins.
8. Resuspend the cells in st. ice-cold 0.1 M CaCl2 by gently flicking the tube and incubate
for 20 mins in ice.
9. Centrifuge at 2500 rpm/ 4°C/ 10 mins. Discard the supernatant.
10. Suspend the cells in 100 µl of st. ice-cold 0.1 M CaCl2.

1. Transformation Expression In Host Cell

Materials

 E.coli competent cells stored at 4°C.

 DNA insert/plasmid.
 Sterile Luria Bertini broth
 St. LB Agar plates with antibiotic
 37°C shaker incubator
 42°C water bath
 Ice buckets with ice
 Pre chilled st. Eppendorf tubes
 37°C incubator
 St. Micro tips (10µl, 200µl, 1000µl)
 Micropipettes

Preparation of Antibiotic: (100 mg/ml)

1 g of Ampicillin sodium salt + 10 ml of st. Distilled water, mix it

Filter sterilise through 0.2 µm filter

Aliquot 500 µl in st. 1.5 ml Eppendorf and store it at -20°C

The final concentration of antibiotic into the media should be 100µg/ml from the stock of 100
mg/ml.

LB media

1 ml

5 ml
15 ml

50 ml

500 ml

Ampicillin

(100 mg/ml)

1 µl/ml

5 µl/ml

15 µl/ml

50 µl/ml

500 µl/ml

Protocol

1. Thaw a tube of E.coli competent cells in ice.

2. Resuspend the cells by flicking tube gently, and then remove 100 µl in prechilled 1.5 ml
tube.
3. Add 200-500 ng of DNA (volume not greater than 10 µl).
4. Flick the tube slowly
5. Immediately incubate the tubes in ice for 30 mins.
6. Heat-shock the cells for 2 minutes in water bath at 42°C, don't shake.
7. Immediately place the tubes on ice for 2 mins.
8. Add 900 µl of st. LB broth and incubate for 1 hour at 37°C / 100 rpm.
9. Add 50 µl of transformation mixture to first st. LB agar plate containing antibiotic.
Spread it with st. spreader.
10. Add 100 µl to second st.LB agar plate containing antibiotic. Spread with st. spreader.
11. Keep the plates at RT for drying (15 mins).
12. Incubate the plate at 37°C overnight.

1. IPTG Induction

Isopropyl β-D-1-thiogalactopyranoside induces lac operon and recombinant gene expression in

E. coli as follows:

The lac operon is one of the most commonly used systems for creating recombinant proteins in
E. coli that can then be purified and studied. Usually your gene of interest is interested into a
commercial vector (pET vectors are common) that contains:

1. A gene coding for antibiotic resistance.

 2. The LacI gene from the lac operon that codes for the lac repressor (LacI).
3. Your gene of interest just after the T7 promoter DNA sequence, the lac operator DNA
sequence, and the ribosome binding site.

Chemicals and reagents:

1. 1 M stock of filter sterilised IPTG.

2. E. coli culture with plasmid having fusion protein gene.
3. Sterile LB broth with 50µg/ml Ampicillin & 50µg/ml Chloramphenicol.

Instruments:

1. Colorimeter
2. Centrifuge
3. Incubator shaker at 37°C and 150 rpm

Miscellaneous:

1. Micropipettes and micro tips

2. Sample loading dye
3. Autoclaved distilled water

Preparation of chemicals and reagents:

Preparation of stock solution of 1M Iso-Propyl-Thio_Galactosid (IPTG)

1. Dissolve 0.238g of IPTG in 1 ml of distilled water.

2. Filter sterilise with a 0.22µ syringe filter
3. Store in 1ml aliquots at -20°C.

Method:

1. Take a single colony of E.coli containing desired vector and inoculate in 10ml sterile

Leuria Bertini broth media (Ampicillin + Chloramphenicol) incubate at 37°C overnight.

1. Next morning inoculate 3-5% of O/N culture in a 50ml sterile Leuria Bertini Broth media
(ampicillin + Chloramphenicol) and incubate at 37°C with shaking at 150 rpm.
2. Keep one flask containing 50ml sterile LB media without any inoculums which can use
as blank.
3. Monitor the optical density 620nm till it reaches in between to 0.55 – 0.65.
 4. Take out 1ml of culture in Eppendorf, spin down, discard supernatant, and add 50µl of
50mM Tris (pH 8.0) and 50µl of 4X loading dye boil till cells disrupt, spin again and
store in freezer. This is the uninduced sample.
5. Add 0.5mM/1mM/1.5mM (final concentration) of IPTG from 1M stock, Take out 1ml of
culture and follow the same procedure. This is the zero hour sample.
6. Re incubate remaining cultures at 37°C at shaking with 150rpm, and collect 1ml fractions
after every 1 hour and follow the same procedure for '1st,' 2nd' 3rd' 4th hour samples.
7. Load 10µl of the each sample on 10% SDS-PAGE.
8. Run the gel till dye front reaches 80-90%.
9. Stain the CBB staining solution for 5-7mins, destain with destaining solution for 15mins
for 3 times.
10. Document the gel under Gel Documentation system.

1. SDS-PAGE

(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

Electrophoresis is the study of the movement of charged molecules in an electric field. SDS –
PAGE involves separation of proteins based on their size. The general electrophoresis techniques
cannot be used to measure the molecular weights of the biological molecules because the
mobility of a substance in the gel is influenced by both charge and size. In order to overcome
this, if the biological samples are treated so that they have a uniform charge, electrophoretic
mobility then depends primarily on size. The molecular weight of protein may be estimated if
they are subjected to electrophoresis in the presence of a detergent sodium Dodecyl sulfate and a
reducing agent β-mercaptoethanol. SDS disrupts the secondary, tertiary and quaternary structure
of the protein to produce a linear polypeptide chain coated with negatively charged SDS
molecules. Β-mercaptoethanol assists the protein denaturation by reducing all disulfide bonds.
By heating the sample under denaturing and reducing condition, proteins become unfolded and
coated with SDS detergent molecules, acquiring a high net negative charge i.e. proportional to
the length of the polypeptide chain. When loaded onto a gel matrix and placed in an electric
field, the negatively charged protein molecules migrate toward the positively charged electrode
and are separated by a molecular sieving effect. After visualisation by a protein-specific staining
technique, the size of a protein can be estimated by comparison of its migration distance with
that of standard of known molecular weight.

Chemicals and reagents:

1. 1.5M Tris HCl (pH 8.8)

2. 0.5M Tris HCl (pH 8.0)
3. Acrylamide mixture (30%)
4. TEMED
5. 10% Ammonium Per Sulphate (APS)
6. 10% SDS
 7. 1X TG buffer
8. 50mM Tris (pH 8.0)

Instrument:

1. Centrifuge
2. Powerpack
3. SDS-PAGE apparatus

Miscellaneous:

1. Autoclaved distilled water

2. Sterile micro tips
3. Micropipettes
4. Sample loading dye

Preparation of chemical reagents:

1. 1.5 Tris HCl (pH 8.8) - 250ml

Weigh 59.1gm of Tris HCl add 200ml D/W, dissolve, adjust the pH to 8.8 with 10N NaOH.
Make up the volume up to 250ml.

1. 0.5M Tris HCl (pH 6.8) – 250ml

Weigh 19.7gm of Tris HCl add 200ml D/W, dissolve, adjust the pH to 6.8 with 10N NaOH.
Make up the volume up to 250ml.

1. 1X TG buffer – 200ml

Take 20ml of 10X TG buffer ass 180ml of D/W.

1. 10% SDS -20ml

Take 2gm of SDS and dissolve in 20ml of D/W

1. 10% ammonium per sulphate(APS) – 1ml

Weigh 0.1gm of APS in Eppendorf and add 1ml of D/W.

 1. Acrylamide composition (30% w/v)

Acrylamide

29.22g

Bisacrlyamide

0.78g

D/W

100ml

1. CBB Staining solution composition

CBB stain R250

0.25g

Glacial Acetic Acid

10ml

Methanol

40ml

D/W

50ml

Total Volume

100ml

1. CBB destaining solution composition

Glacial Acetic Acid

10ml

Methanol

40ml

D/W
50ml

Total Volume

100ml

1. Resolving gel composition

Distilled water

4.1ml

1.5M Tris-HCl, pH 8.8

2.5ml

10% (w/v) SDS

50µl

Acrylamide mixture (30% w/v)

3.3ml

10% (w/v) Ammonium per sulphate

50µl

TEMED

7µl

Total Volume

10.007ml

1. Stacking gel composition (4%)

Distilled water

3.075ml

0.5M Tris-HCl, pH 6.8

1.25ml

10% (w/v) SDS

25µl
Acrylamide mixture (30% w/v)

0.67ml

10% (w/v) ammonium per sulphate

25 µl

TEMED

5 µl

Total Volume

4.425ml

Method:

1. Assembling the glass plate.

2. Ensure the glass plates, spacers, combs are clean and dry. The glass plates should be
cleaned with 70% ethanol.
3. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then
place two spacers of equal thickness along the rectangular plate. Next place the shorter
glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are
aligned. Place the clamps on the sides of the plates.
4. Hold the glass plates upright on the workbench and add 2% agar on the bottom and sides
of the glass plates so that it enables proper sealing.

1. Casting the gels.

2. Prepare 10% resolving gel and 5% stacking gel.
3. Prepare the separating gel monomer solution and mix the solution after adding each
reagent by swirling the container gently. Pour the separating gel into the glass plate
assembly.
4. Immediately overlay the monomer solution with 20 – 30 µl of water-saturated n-butanol
which prevents oxidation of gel and easily evaporates.
5. Allow the gel to polymerise for 10 – 15 minutes.
6. Pour the stacking gel into the glass plate assembly.
7. Place a comb into the assembled gel sandwich. Insert the comb at an angle to avoid
trapping bubbles under the comb.

1. Inserting comb into resolving gel.

2. Allow the gel to polymerise for 10 minutes.
3. With a marker pen, mark the teeth of the comb on the glass plate. This is to ensure the
position of the wells.
 4. Remove the comb.
5. Gel is placed in the buffer chamber and running gel buffer is added into the chamber.

1. Loading the samples.

2. Insert the pipette tip to about 1 – 2 mm from the well bottom and load the sample.
3. Ensure there should be no bubbles which may interfere with the separation.

1. Running the gel.

2. Check that the buffer in the upper buffer chamber is full because leakage of the buffer
may occur.
3. Place the lid on top of the tower buffer chamber. Make sure that the connection is correct,
i.e. black to black and red to red.
4. Attach the electrical leads to a suitable power pack with the proper polarity. Run the gel
at 100V. Stop the electrophoresis when the tracker dye is ˵1 cm above the end of the glass
plates.

1. Removing and staining the gel.

2. Remove the gel from the buffer chamber.
3. Loosen all screws of the clamp assembly and remove the glass plate sandwich from it.
4. Push one of the spacers out to the side of the plates without removing it.
5. Gently twist the spacer so that the upper glass plates pulls away from the gel.
6. Cut the gel on one side (to orientate the gel)
7. Remove the gel by gently grasping two corners of the gel and place it in the container
containing Coomassie brilliant blue stain. Make sure that the gel is fully submerged in the
staining solution.
8. Stain the gel for 5 – 7 minutes, and keep it in gel rocker.
9. Destain the gel in a destaining solution 3 times for 15minutes or till bands are visualised.
10. Approximately determine the molecular weight of the visualised protein bands by
comparing them with the molecular weight markers.
Chapter 5: Results and Discussions

1. gDNA isolation

Lane 1 Lane2 Lane3

Lane4 Lane5 Lane6 gDNA isolated from E.coli

Figure 1 shows agarose gel electrophoresis of bacterial gDNA loaded on 0.8% gel of agarose gel
stained with EtBr

Lane 1 shows 1 kb ladder

Lane 2-6 shows isolated genomic DNA of bacteria E.coli

1. For plasmid isolation:

Lane 1 2

345

6
Figure 2 shows agarose gel electrophoresis of pET21b plasmid isolation sample loaded on 1%
gel of agarose gel stained with EtBr

Lane 1 shows 1 kb ladder

Lane 2-6 shows the isolated plasmid sample

1. For PCR

12

345

6 amplified PCR product

Figure 3 shows agarose gel electrophoresis of PCR product loaded on 1.5% gel of agarose gel
stained with EtBr

Lane 1 shows 1 kb ladder

Lane 2-6 shows amplified PCR product

 1. For Restriction Enzyme digestion

12

345

Figure 4 shows agarose gel electrophoresis of restriction enzyme loaded on 1% gel of agarose
gel stained with EtBr

Lane 1 shows 1 kb ladder

Lane 2-6 shows the restriction enzyme digestion of EcoRI and EcoRV

1. IPTG SDS-PAGE

12
345

67

Figure 5 shows SDS-PAGE analysis of induction samples of protein eGFP

Lane 1 shows protein ladder

Lane 1, 2, 3, 5, 6, 7 show the induction of protein at different hours

Chapter 6: Conclusion

The general aim of cloning is to express specific proteins in vitro in order to study genes and
genes manipulation effects was performed and studied. Gene cloning was carried out according
to the following steps such as choice of host organism i.e. gene of interest (GOI) into a cloning
vector, preparation of vector DNA, preparation of DNA to be cloned, creation of recombinant
DNA with ligase, introduction of recombinant DNA into host organism, selection of organism
containing vector sequence, screening for clones with desired DNA inserts and biological
properties.

The plasmid used is pET21b in XL Blue Strain of E.coli bacteria. We got the amplified plasmid
which was further isolated using PCR technique. The amplified and isolated plasmid was cut
using restriction enzymes and further ligated to obtain the required gene viz. eGFP which was
incorporated into competent cells n transformed to get copies of the same.

Thus the molecular biology technique of gene cloning was efficiently carried out and the aim of
getting multiple copies of a single DNA molecule starting from a single living prokaryotic cell to
generate a large population of cells containing identical DNA molecules was achieved.

Chapter 7: References

1. Modern Genetic Analysis, Griffiths, et.al W.H. Freeman & Co. NY Chapter 10, 1999

1. DNA Extraction- CTAB Method, Worden Lab prepared by Alexandra Worden, 14 March
2009
1. Gene Cloning and DNA Analysis: An Introduction (Brown, Gene Cloning and DNA
Analysis) by T. A. Brown

1. QIAprep Miniprep Handbook, January 1999

1. Molecular Biology, Weaver, RF, 2nd edition. McGraw Hill, San Francisco. p.p. 93-94,
2002

1. Principles of nucleic acid separation by agarose gel electrophoresis. Muhittin Yilmaz

1. Mullis, K., The unusual origin of the polymerase chain reaction. Scientific American,
April p.p. 56-65 1990