UBS2210

Genetic Engineering Lab

Faculty of Biosciences
Institute of Biosciences & Technology

BSc (H) Biotechnology

First year
LAB MANUAL
of
Genetic Engineering Lab (UBS2210)

Dr. Pratibha Gupta

Dr. Sankalp Misra
Er. Ayushi Verma
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List of Experiments
S.No. Name of the Experiment
1. To demonstrate the basic of genetic engineering experiment.
2. To perform cDNA synthesis.
3. To perform restriction digestion of plasmid DNA.
4. To perform ligation of DNA molecules.
5. To prepare bacterial competent cells using CaCl2.
6. To check the competency of competent cells.
7. To demonstrate the transformation of E. coli cells.
8. To perform PCR to amplify a DNA fragment.
9. To demonstrate Hershey and Chase experiment.
10. To perform bacterial conjugation.
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EXPERIMENT-1
Aim: To demonstrate the basic of genetic engineering experiment.

Introduction: Tools and techniques of genetic engineering:

Genetic engineering is the process of using gene cloning and other genetic manipulations to
isolate specific genes and use it for research and other purposes. Each DNA strand contains
thousands of genes. Gene cloning is the process of replicating a specific set of genes from a strand
of DNA.

Important Molecular Tools In Genetic Engineering

Here is a list of a genetic engineer’s molecular tools/enzymes most commonly used in genetic
engineering experiments:

1. Polymerase Chain Reaction (PCR)

Fig 1. PCR machine

Polymerase Chain Reaction (PCR) is the process of replicating multiple copies of the genes of
interest. The discovery of thermostable DNA polymerases, such as Taq Polymerase, has made
it possible to manipulate DNA replication in the laboratory. It amplifies the quantities of DNA
segments. Primers areused to identify the gene of interest and replicate them. These copies can
then be separated and purified using gel electrophoresis.
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2. Restriction Enzymes (Molecular Scissor)

The discovery of enzymes known as restriction endonucleases has been essential to protein
engineering. Based on the nucleotide sequence, these enzymes cut DNA at specific locations.
DNA cut with a restriction enzyme produces many smaller fragments of varying sizes. These can
be separated using gel electrophoresis or chromatography.

3. Gel Electrophoresis

Purifying DNA from cell culture, or cutting it using restriction enzymes wouldn’t be of much use
if we couldn’t visualize the DNA. Gel electrophoresis helps visualize the size and type of DNA
extracted using PCR and restriction enzymes. It is also used to detect DNA inserts and knockouts.
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4. DNA Ligase

DNA ligase can create covalent bonds between nucleotide chains. This is done to create
recombinant strands or close a circular strand that has been cut by restriction enzymes. The
enzymes DNA polymerase I and polynucleotide kinase are also important for filling in gaps or
phosphorylating the 5′ ends, respectively.

5. Plasmids

Plasmids are small, circular pieces of DNA that are not part of the bacterial genome but are
capable of self-replication. It is used as vectors to transport genes between microorganisms. Once
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the gene of interest has been amplified with PCR, the gene and plasmid are cut by restriction
enzymes and ligated together.
The resulting combination is known as Recombinant DNA. Viral (bacteriophage) DNA can also
be used as a vector, as can cosmids, which are recombinant plasmids containing bacteriophage
genes.

6. Transformation/Transduction

Transformation is the process of transferring genetic material in a vector, such as a plasmid,

into host cells. The host cells are exposed to an environmental change, such as electroporation,
which makes them “competent” or temporarily permeable to the vector. The larger the plasmid,
the lower the efficiency with which it is taken up by cells.
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Larger DNA segments are more easily cloned using bacteriophage, retrovirus, or other viral
vectors or cosmids in a method called Transduction. Phage or viral vectors are often used in
regenerative medicine but may cause the insertion of DNA in parts of our chromosomes where
we don’t want it, causing complications and even cancer.

7. Identification of Transgenic Organisms

Not all cells will take up DNA during transformation. Therefore, it is essential to identify the cells
that undergo a transformation and those that have not. Generally, plasmids carry genes for
antibiotic resistance, and transgenic cells can be selected based on the expression of those genes
and their ability to grow on media containing that antibiotic. Alternative methods of selection
depend on the presence of other reporter proteins such as the x-gal/lacZ system, or green
fluorescence protein, which allow selection based on color and fluorescence, respectively.
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EXPERIMENT - 2
Aim: To perform cDNA synthesis.
Principle: The synthesis of DNA from an RNA template, via reverse transcription, produces
complementary DNA (cDNA). Reverse transcriptase (RTs) uses an RNA template and a short
primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA,
which can be used directly as a template for the Polymerase Chain Reaction (PCR). This
combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance
RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning
of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA
Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without
amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is
required.

Reagents:
i) 5X First Strand Buffer
ii) 10mM dNTP Set
iii) 0.1M DTT
iv) Random Primers
v) RNaseOUT Ribonuclease Inhibitor
vi) SuperScript RNase H- Reverse Transcriptase

Procedure:
1. Combine 3-5 μg total RNA and molecular grade water to 8 μl final volume.
2. Add 3 μl Random Primers.
3. Add 1 μl dNTP mix.
4. Vortex and then spin down tube.
5. Incubate at 65°C for 5 min.
6. Place tube on ice.
7. Add 4 μl 5X Buffer, 2 μl DTT and 1μl RNAseOut.
8. Vortex and then spin down tube.
9. Incubate at 42°C for 1 min.
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10. Add 1μl SuperScript II RNase H- Reverse Transcriptase.

11. Incubate at 42°C for 60 min.
12. Incubate at 70°C for 15 min.
13. Add 180 μl molecular grade water.

NOTES
10 mM dNTP Mix contains:
10 μl dATP [100mM]
10 μl dCTP [100mM]
10 μl dGTP [100mM]
10 μl dTTP [100mM] in 60 μl molecular grade water

Results and Observations:

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EXPERIMENT - 3
AIM: To perform restriction enzyme digestion.

Why we need to perform restriction digestion?

Cloning has become a very common mode to manipulate gene sequences in Biotechnology.
Restriction digestion is one of the very common methods to generate DNA sequence of interest.
Restriction digestion method is used to generate target DNA as well as to cut vector DNA. Once
these ends are produced both in target DNA as well as in vector, then DNA ligases can be used to
insert target DNA into the vector. Restriction enzyme digestion is also used for various methods for
DNA typing like RFLP etc. After restriction digestion the banding pattern of DNA fragments can
be analyzed by agarose gel electrophoresis so that DNA typing of the organisms can be either
performed or to be established.

Principle:

It has been established that various restriction enzymes can be used for restriction digestion. Every
restriction enzyme cuts or digests DNA at specific site, which is known as restriction site. That is
the reason that after restriction digestion each organism shows a unique pattern. It is used as
restriction digestion pattern. When a bacterial DNA is treated with restriction enzyme, due to site
specific activity of restriction enzyme, bacterial DNA is cleaved into smaller fragments. This
cleavage is dependent upon the presence of those specific restriction sites as well as numbers of
those specific sites present in DNA. After electrophoresis of restriction digestion can be observed
for a specific DNA. When genomic DNA is exposed to restriction enzymes, due to larger size of
genome, so many fragments are generated. This is the reason it is difficult or impossible to find a
specific restriction digestion pattern over agarose gel electrophoresis. Below mentioned figure
depicts restriction sites in DNA for EcoRI.

↓
G AATT C

C TTAA ↑ G
Restriction sites for EcoRI

STEPS:

1. Preparation of reaction mixture:

The reaction mixture is prepared by addition of DNA with the optimal concentration of
restriction enzymes along with specific restriction buffer. After addition of all the
components, this mixture is incubated to initiate and complete the digestion of DNA
template.
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2. Agarose gel electrophoresis:

After completion of digestion the banding pattern of digested DNA is analyzed by agarose
gel electrophoresis. During electrophoresis Hind III markers and suitable negative control
are used for interpretation of result.

MATERIALS:

Isolated DNA, 10x Restriction buffer, EcoRI (10-20 units/µl), Distilled water, Ice, Eppendorf tubes,
Micropipette, Micropipette tips, Water bath, and Heating blocks etc.

METHOD:

A. Restriction Digestion:
1. Take two eppendorf tubes and mark one with “Test” and the other tube as “Control”.
2. Prepare reaction master mix as mentioned in Table.

Items Sample N * Sample

10x Buffer 5 µl
EcoRI 1 µl
Distilled Water 24 µl
Total 30 µl
N*; N is the number of samples +2. if you have to digest 4 samples then prepare reaction mixture for
at least 6 samples, as the volumes are too less. So you may not be able to pipette the last few drops.

3. Prepare reaction tube as given in Table.

Master mix DNA DW
Control 30 µl - 20 µl
Test 30 µl 20 µl -
4. Mix contents of tubes by up and down movement of pipette. Briefly centrifuge for 5
sec at 1000 rpm.
5. Incubate tubes at 37˚C, over heating block (or water bath) for one hour. (Note:
Samples can also be left over night at 37˚C).
6. If no other manipulation is required in that case to stop reaction by adding 100 µl of
stop solution in 50 µl of reaction mixture.
7. Evaluate result over agarose gel electrophoresis.

OBSERVATIONS:
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We have digested the bacterial DNA with EcoRI enzyme. It has

given……………………………fragments of DNA, which range from………………………kb
to……………kb.

B. For Agarose Gel Electrophoresis:

Sample Name Sample volume Loading Buffer

(10 µl) (2 µl)
1
2
3
4

RESULTS:

The bacterial DNA was digested with EcoRI and it has given
……………………………..fragments.

CNCLUSIONS:

The DNA has been successfully digested by EcoRI restriction enzyme which is confirmed by the
distinct band pattern on agarose gel electrophoresis.

PRECAUTIONS:

1. All the steps should be carried out on ice.

2. Store digested samples in freeze until further use.
3. Always wear gloves.
4. DNA should be free of contaminants.
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EXPERIMENT – 4
AIM: To perform DNA ligation.

Introduction: After restriction digestion either sticky ends or blunt ends DNA fragments are
produced. Similarly restriction digestion can produce sticky end or blunt ends even in case of
vectors, too. DNA fragment/s can be inserted into vector. Insertion or joining of DNA fragments
occurs due to DNA fragments occurs due to DNA ligation. This method has numerous applications
for molecular biology and recombinant DNA technology. Insertion of new DNA (or gene of interest)
is possible due to the process known as DNA ligation. This method has proven to be an excellent
tool for gene cloning, and gene cloning has numerous applications.

Principle: DNA ligation is the process of two DNA fragments. This DNA fragment could be either
of same DNA or from two different sources. Ligation process involves creation of phospho-di-ester
bond between 3’ hydroxyl of one nucleotide and 5’ phosphate of another nucleotide. Ligation
reaction is usually catalyzed by DNA ligase. Ligation occurs between DNA fragments either with
blunt ends or sticky ends. Ligation of sticky ends with blunt ends or sticky ends. Ligation of sticky
ends DNA is comparatively easier compared to blunt ends DNA. T4 DNA ligase is the most
commonly used enzyme for DNA ligation. T4 DNA ligase can ligate both “sticky ends” and “blunt
ends” DNA fragments. Agarose electrophoresis picture of ligated DNA is shown in Fig- MB-15.

Steps:

1. Restriction Digestion: Perform restriction digestion to produce DNA fragment to be inserted into
vector. Similarly cut vector with the same restriction enzyme which has been used to do digestion
of DNA.
2. DNA ligation: In this step DNA fragment/s either plasmid DNA or vector DNA along with T4
DNA ligase and ligation buffer is incubated to complete the process of DNA ligation.
3. Confirm DNA ligation by running sample over agarose gel electrophoresis.

Materials: T4 DNA ligase, 10x T4 DNA ligase buffer, DW, Linearized vector, DNA fragment/
insert, etc.

Method:

(a) Restriction digestion: Follow the protocol for restriction digestion.

(b) DNA Ligation:
i) Collect digested DNA and vector.
ii) Mix 10ng of DNA insert with 50ng of linearized vector. It should be in 1:5 ratio.
iii) Prepare ligation reaction in total volume of 10µ.
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iv) Incubate at 26°C for 60min for cohesive ends and 14C for 16hr for blunt ends.

(c) Agarose Electrophoresis: Perform agarose gel electrophoresis of DNA.

(d) Transformation: Follow the protocol for transformation as provided in this section of the book.

Ligation Reaction Mixture:

Components For one reaction For ‘N’ reaction

Digested vector (50ng)
Insert
Sterile water
10x ligation buffer
T4 DNA ligase
TOTAL

N: N+1 sample

OBSERVATIONS

DNA Conc. (in ng)

Vector Conc. (in ng)

Enzyme (Volume)

Incubation Time

Incubation Temp

Table OT-MB-15: Observations for DNA Ligation.

Sample Ligation
1
2
3
4
+ for ligation and – for no ligation
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RESULTS

The agarose gel electrophoresis and transformation results confirm the ligation of DNA into vector.

CONCLUSION

The digested DNA fragment is successfully inserted into the vector which has been transformed to
the desired competent cell.

PRECAUTION

1. Purify the DNA properly before going to ligation step.

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EXPERIMENT-5
AIM- To prepare bacterial competent cells.

INTRODUCTION-

In any transformation experiments, DNA has to enter into the bacterial cell.The bacterial membrane
is the main barrier for the entry of foreign DNA into bacteria. Even though plasma membrane is
selectively permeable, but plasma membrane does not allow DNA, we need to create transient pores
in bacterial cells are competent for the entry of foreign DNA, therefore these bacterial cells are called
as ‘’competent cells’’. Competent cells are in common use in laboratories dealing with gene cloning
or expansion of foreign DNA, where bacteria (competent cells) act as host cells.

PRINCIPLE –

Natural competence in bacteria occurs through 3 different methods i.e., conjugation, transformation
and transduction. Bacterial cells have to be made artificially competent so that bacterial cells can
become permeable to foreign DNA, making bacterial cells competent means to make bacterial
plasma membrane permeable for the entry of DNA. Bacterial surface is negatively charged as well
as DNA is also negatively charged, since bacteria and DNA both has similar charges, therefore it is
difficult for DNA to enter in to bacterial cells. To promote DNA entry, divalent cations are used in
the process and these divalent ions plays a crucial role by shielding charges by coordinating
phosphate groups and other negative charges. Application of divalent ions changes electrical charge
on the bacterial cell surface and weaken bacterial cell membrane. The final end result of this process
supports entry of foreign DNA into bacterial cells, because these cells are competent enough to
allow entry of foreign DNA.

STEPS-

1. Growing of bacterial cells

The first step is to grow bacteria up to the 0.2 – 0.5 OD at 600nm in LB Broth. At this stage
bacteria in the culture are in log phase.

2. Chemical treatment
At this step bacterium is treated with CaCl2 solution. Treatment of bacteria with CaCl2 increases
permeability of bacterial plasma membrane.

MATERIALS-

Bacteria, Spectrophotometer, LB agar plate, LB broth, Glycerol, CaCl2, Tubes, Centrifuge, etc.
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METHOD-

1. Grow E. coli in 5ml LB broth overnight.

2. Take 2.5ml of overnight grown bacteria and grow in LB broth at 37C over a shaker. (Ideally it
is good to dilute the overnight culture by 1:100 dilution ,then grow bacteria in 250ml of LB in 1
Lit conical flask)
3. Check OD of culture at 600nm till OD reaches to 0 .25-0.30. (It takes ~1.5-2.0 hrs.). You should
aim to dilute the overnight culture by 100 fold. That means mix 1ml of bacterial culture to 100ml
of LB broth.
4. Transfer culture into 50ml centrifuge tubes .Now onwards all steps have to be done in cold.
5. Centrifuge tubes at 3,000rpm 10min at 4C.
6. Discard supernatant. This can be done by pouring of the media. The residual media can be
removed by Pasture pipette or microliter pipette.
7. Re-suspend cell in 30ml of chilled 100mM CaCl2 for 30 min on ice and shake the bacterial
suspension with hand intermittently.
8. Centrifuge cells at 3000rpm for 10min at 4C.
9. Discard supernatant.
10. Re-suspend bacterial pallet in 5 to 10ml of 100mM CaCl2 plus 15% Glycerol.
11. Prepare 100μl aliquots of this bacterial cell suspension and store at -70C

OBSERVATION-

We have prepared competent cells and those cells are frozen at -70C till further use.

RESULT-

Competent cells are prepared successfully and are stored at -70C for further use.

CONCLUSION –

In the process of chemical treatment to the cell in use to prepare competent cells which is an efficient
and cost –effective method.

PRECAUTION_

• All procedure should be done cold condition.

• After CaCl2 treatment bacterial cell becomes quit fragile, so do not vortex them.
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EXPERIMENT-6

Aim: To perform Agarose Gel Electrophoresis

Introduction:

Agarose gel electrophoresis is a method for separation (by size), quantifying, and purification of
nucleic acid fragments mixture, and analysis of DNA restriction fragments. It is one of the most
widely used techniques in biochemistry and molecular biology. Agarose is a liner polymer
composed of alternative residues of D-galactose and 3,6-anhydro-L-galactopyranose joined by α
(1→3) and β (1→4) glycosidic linkages. Agarose and acrylamide matrices are used to separate DNA
by gel electrophoresis. The choice of gel matrices and gel concentration depends on the size of
nuclear acid molecules, as the concentration of the agarose or acrylamide determines the size of the
pore:

w/v % Gel type Size of DNA fragments (Kb = 1000 bp)

0.5 % 1 kb to 30 kb
0.7 % 800 bp to 12 kb
1.0 % 500 bp to 10 kb
1.2 % 400 bp to 7 kb
1.5 % 200 bp to 3 kb
2.0 % 50 bp to 2 kb

Under physiological conditions, DNA is a negatively charged molecule due to the presence of
phosphate groups in the backbone. Therefore, in aqueous media, under the influence of an electrical
field, DNA molecules will move through an agarose matrix towards the positively charged anode,
at a rate that is inversely proportional to the molecular weight. The electrophoretic migration rate of
DNA through agarose gel depends on the following: size of DNA molecules, the concentration of
agarose gel, the voltage applied, the conformation of DNA, and the buffer used for electrophoresis.

Several buffers are used for agarose gel electrophoresis, but the most common are Tris-acetate
EDTA buffer (TAE) and Tris-borate EDTA buffer (TBE). The DNA mobility in the TBE buffer is
approximately two times slower than in the TAE buffer. This is due to the lower porosity of agarose
gel when agarose polymerizes in the presence of borate.

Since DNA is colorless, the loaded sample needs to be tracked. This is achieved by using a loading
dye solution. Finally, to visualize DNA (result), agarose gels are usually stained with ethidium
bromide and illuminated with UV light. Identifying the size of a DNA sample is one of the common
AGE uses and this is accomplished through what is called: a DNA marker (Ladder). DNA and RNA
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size markers contain a mixture of DNA (or RNA) fragments of known length, making them suitable
for estimating the fragment length of concurrently run samples.

Principle: Nucleic acids are separated by applying an electric field, so these negatively charged
molecules will move through an agarose matrix towards the anode, and the biomolecules are
separated by size in the agarose gel matrix, where the distance traveled by a DNA molecule is
inversely correlated with its size.

Materials: Agarose powder, 1X TBE buffer (89 mM Tris-base, 89 mM boric acid, and 2 mM
EDTA) prepared from 10X TBE, Ethidium Bromide (5 mg/ml), Gel loading dye (Glycerol and
orange dye), 1 kb and 100 bp DNA ladder, horizontal electrophoresis apparatus and power supply.

Protocol:

1. Measure the desired grams of agarose to make 1% agarose gel.

2. Heat the solution to boiling in the microwave to dissolve the agarose to produce a homogeneous
mixture.

3. Add 4 µl of ethidium bromide CARFULLY to the dissolved agarose and mix.

4. Get a gel plate and a comb. Put the two dams into the slots on each side of the gel plate. Make
sure that they fit tight. Pour the melted agarose onto the gel plate in the electrophoresis tray.

5. Place the comb in its place. Let the gel cool to room temperature.

6. Place the gel in the electrophoresis chamber.

7. Pour enough electrophoresis buffer (1X TBE) to cover the gel to prevent overheating of the gel.

8. Carefully remove the comb.

9. Prepare the DNA sample by mixing around 300 ng of DNA sample with 3-4 µl of loading dye.

10. Add 3 µl DNA ladder into the first well by using a micropipette.

11. Carefully place the prepared samples into adjacent wells.

12. Electrophorese the samples at 95 V for 45 minutes. (Check the gel while it is running).

13. Carefully remove the gel, place it onto the UV light box, and take a picture of the gel.

Results: Picture of the gel.

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EXPERIMENT-7

Aim: To amplify DNA by Polymerase Chain Reaction (PCR)

Introduction: PCR is a method used to amplify a gene sequence to generate multiple copies of same
sequence. PCR reaction is possible due to the discovery of Taq Polymerase enzyme. This enzyme
is used in PCR as it can sustain variations of temperature during thermal cycling. PCR produces
specific product due to use of specific primers. Primers support amplification of only primer specific
sequence. PCR has become a versatile technique because of numerous modifications at various steps
due to versatility of PCR. It has become a useful method for various purposes like diagnosis,
mutation, detection, cloning, etc. PCR can be used in forensic science, anthropological investigation,
archaeological investigations, etc.

Principle
PCR requires two oligonucleotide primers, which flank DNA sequence that has to be amplified. In
the first step target DNA is denatured. Next primers hybridize to opposite strands of DNA and
orientated in such a manner that DNA synthesis can occur between two primers. The extension step
during PCR leads to create two double stranded target regions. This newly synthesized DNA again
gets denatured and is ready for second cycle of DNA hybridization and extension, repeated cycling.

Steps

1. Preparation of master mixture

In this step various components required for PCR reaction are mixed in one tube which is called as
master mix. Master Mix does not contain DNA template which has to be amplified. The main
purpose for the preparation of master mix is to provide homogenous mixture of various PCR
components for each sample. This assures consistency of amplification reaction across different
DNA samples.

2. Thermal cycling
DNA, after being added to master mix has to undergo thermal cycling. Conditions of thermal cycling
vary for primer set/s as well as vary from thermal cycler to thermal cycler. These steps have to be
empirically determined.

3. Conformation of PCR product

After the completion of PCR, the final amplified product has to be confirmed by agarose gel
electrophoresis.
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Reagents and equipments:

i. Target DNA,
ii. 10x PCR reaction buffer
iii. MgCl2,
iv. dNTPs,
v. Primer,
vi. Taq DNA polymerase,
vii. Distilled Water,
viii. PCR tubes,
ix. thermal cycler,
x. microliter pipettes, pipette tips, etc.

METHOD:

A. PREPARATION OF MASTER MIX AND REACTION MIXTURE

1. Thaw PCR reagents on ice.
2. Prepare master mixture on ice. Follow the details given in table.
3. Mix master mix by tapping tubs with fingers.
4. Centrifuge at 1000rpm for 10- 15 sec.
5. Aliquot master mix (40µl) in PCR tubes. ( label tubes before adding master mix)
6. Add 10µl of template DNA to each tube.
7. Mix contents by tapping tube with hands.
8. Centrifuge PCR tubes at 1000rpm for 10 sec.

B. Amount of master mix preparation

Components For 1 reaction For N*reaction

MgCl2 8µl
PCR buffer(5x) 10µl
dNTP mix (10Mm) 1µl
upstream primer 1µl
Taq 0.5µl
Nuclease free water 19.5µl
Total 40µl

*; N+2 ARE TOTAL NO. OF SAMPLES

C. Reaction set up.

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Components master mix DW DNA

(40µl) (10µl) (10µL)

Negative control + + -

Positive control + - +

Sample + - +

D. THERMAL CYCLING FOR PCR

1. Turn on the thermal cycler.

2. Step up the thermal cycler for following conditions.
3. Keep tubes in the slot of PCR machine
4. Start thermal cycling.
Table: Conditions for thermal cycling.

Step Temperature Duration

Inactivation 96°C 5 min.

Deactivation 95°C 15 sec

Annealing 49°C 30 sec

Extension 72°C 1 min.

Cycle 30-35

Final extension 72C 10 min.

End of PCR 4C ∞

(E) CONFIRMATION OF PCR PRODUCT

Run agarose gel electrophoresis for the PCR products.

OBSERVATION
Table OT- MB-8: Observations for polymerase chain reaction after agarose electrophoresis of DNA
samples.

Sample Band
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1.

2.

3.

4.

Record observation as ‘+’ or ‘-‘ and comparative intensity

RESULTS
Agarose gel electrophoresis of PCR products confirms single band of PCR product with
expected size of __ bp.
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EXPERIMENT- 8
AIM-To perform bacterial Transformation.

INTRODUCTION –

Bacterial transformation is the procedure where the naked piece of DNA can be transferred into the
bacteria. Transfer of DNA occurs due to weekend cell wall of bacteria. Competent cells are
commonly used for the uptake of foreign DNA (all gene of interest). Chemical treatment is applied
to bacterial to weaken cell wall. This method is conveniently used to produce large member of copies
of same gene which is possible due to rapid replication of bacteria.

PRINCIPLE-

Transformation can cause the genetic alteration in bacterial cell due to direct uptake and
incorporation of exogenous DNA (Gene of interest) from the surrounding .Uptake of genetic
material occurs through bacterial cell membrane .Uptake of genetic material through cell membrane
was first described by British bacteriologist Frederic Griffith . Transformation may occur in nature
in some bacteria but in laboratory condition, transformation experiment are done by using competent
cell .Transformation can be done by using plasmids , cosmids or fragments of genomic DNA.
Advancement and better understanding of this method is the reason of rapid growth of production
of various recombinant products.

STEPS-

1. Transformation-
Give heat shock at 42C for 30 sec to the suspension of competent cells and DNA. Heat shock
facilitates transformation by allowing the entry of large amount of DNA into competent cells.
DNA uptake by competent cells occurs due to opening of pores present on the cell membrane
of competent cells.

2. Screening of transformants-
Screening of transformants from the non-transformed bacterial cells is done by growing
transformed competent cells on agar plate in the presence of specific antibiotics and if
construct has β-galactosidase gene which can help in selection, in that case color of colony
can be visualized.

Materials-

42C water bath, Ice, LB agar plates, Antibiotic, Competent cells, (DH5α), Sterile SOC, Tubes,
Flasks, etc.
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METHOD-

1. Thaw competent DH5α cells on ice.

2. Gently mix cells.
3. Aliquot 50 μl of competent cells for each transformation into 1.5ml tubes that have been pre
–chilled on ice.
4. Add 1-5μl DNA (1-100ng) to competent cells and mix gently by gently tapping tubes with
finger.
5. Incubate DNA with competent cells on ice for 30min.
6. Heat shock at 42C for exactly 30sec without shaking.
7. Incubate on ice for 2min.
8. Add 250μl of pre –warmed (37C) SOC to tubes after completion of transformation.
9. Shake tubes at 37C for 1hr.
10. Spread 20μl and 200μl of each tube used for transformation onto LB plates with appropriate
antibiotics.
11. Incubate LB plates at 37C in inverted position for 24hr.

OBSERVATION-

We have performed bacterial transformation using blue white colony selection method. Presence of
white colonies suggests that transformation was successful.

RESULTS-

Plates are showing blue and white color colonies presences of white colonies suggest that
transformation was successful.

CONCLUSIONS-

A presence of white colony in agar plate suggests that transformation was successful.

PRECAUTIONS-

1. Thaw cells on ice.

2. DO NOT thaw by warming tubes with your hands.
3. To maintain competency, bacteria should be kept on ice at all times .Once they have warmed
up, they are no longer competent and will not take up DNA.
4. Add LB or SOC using sterile procedures in the hood.
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EXPERIMENT: 9

Aim: To perform the Bacterial Transduction.

Introduction:
Transduction is another means of transfer genetic material from one bacterial cell to another. It is a
well-known fact that the transfer of genetic material is essential and useful for the evolution.
Bacterial transduction is one of the means to transfer genetic material via bacterial viruses of
bacteriophages. Transduction is especially important because they explain reasons for
ineffectiveness of antibiotics due to transfer of resistant genes among bacteria growing together,
which happens in natural conditions during infection. In addition, transduction is an important
method for the transfer of gene of interest from one bacterium to another with the help of viruses.
Transduction is one of the probable hopes on the horizon to develop cure or therapies (genetic) for
some of the deadly diseases by inducing genetic modification.

Principle:
Bacterial viruses or bacteriophages acts as an agent/vehicle to transfer genetic material. As a result
of transduction phenomenon bacteriophages infects host bacterium, incorporates genome of
bacteriophage into bacterial genome. Normally after generating sufficient copies of itself
bacteriophage kill the host cell by lysis. Sometimes, the bacteriophage may not lyse the bacterial
cells. Then the incorporated gene/genome also replicates along with the replication of
bacteriophages in host bacteria and it transferred to the bacterial progeny. As a result of bacterial
lysis, bacteriophages carrying new genome/genes of bacterial origin are released in the environment
and newly released bacteriophages infect other bacterial cell. Infection of bacteriophages to new
bacteria releases bacterial gene (acquired due to transduction) to new bacterial cells. During this
process the bacteriophage carries a chunk of the old host’s DNA to the new host.

Steps:
1. Grow bacteria
The first step is to grow bacteria overnight in broth. Overnight grown bacteria is either in log
phase/or late log phase.

2. Grow the culture of bacteriophage

Grow bacteriophages in specific bacterial host so that bacteriophages can be used for transduction.

3. Initiation of transduction
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Mix the bacterial culture with bacteriophages. After completion of bacteriophages infection mix
bacteriophage infected bacteria with other bacteria.

4. Evaluation of transduction
After completion of transduction process, bacteria have to be grown in agar plate in the presence of
specific antibodies.

Material:
E. coli DH5α with streptomycin resistant plasmid (host), E. coli DH5α with kanamycin resistant
plasmid (recipient), M13 bacteriophage culture, LB medium, Kanamycin, Streptomycin, Spreader,
Petriplates, Tubes, etc.

Method:
(A) Transduction Experiment

1. Grow E. coli (DH5α) in the presence of streptomycin in LB broth. (Name as culture-1)

2. Grow M13 bacteriophage in culture.
3. Mix culture-1 with M13 bacteriophage for 4hr at 37˚C in a shaker incubator at slow rotation.
4. Grow overnight culture of E. coli (DH5α) in the presence of kanamycin because it is
kanamycin resistant (Name as culture-2)
5. Incubate culture-1 and cultute-2 together for 4 hour or overnight at 37˚C in a shaker incubator
at 200rpm.

(B) Confirmation of Transduction

After transduction, bacteria have to be grown in the presence of different antibiotics, so that
transfer of genetic information due to transduction can be confirmed.

1. Prepare three different LB agar plates. Add antibiotics in those plates

No Antibiotics, Streptomycin, Kanamycin and Streptomycin + Kanamycin.

2. Mark the plates and divide plates into 3 sections at back with permanent marker and mark as
culture 1, 2 and (1+2).
3. Take a loop of culture and spread on the surface of the plate into their respective section for
24hrand 28hr.
Plate-1: LB Agar

Plate-2: LB Agar with streptomycin

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Plate-3: LB Agar with kanamycin

Plate-4: LB Agar with streptomycin and kanamycin

Observation:
Table-1: Observation for the colonies observed after the 24hr of incubation for bacterial
transduction.

Sample LB LB + LB + LB +Streptomycin and

Streptomycin Kanamycin Kanamycin

{E. coli
(DH5α) with
Streptomycin}

{E. coli
(DH5α) with
Kanamycin}

After
transduction

Table-2: Observation for the colonies observed after 48hr of incubation for bacterial transduction.

Sample LB LB + LB + LB + Streptomycin and

Streptomycin Kanamycin Kanamycin

{E. coli
(DH5α) with
Streptomycin}

{E. coli
(DH5α) with
Kanamycin}

After
transduction
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Result:
We have seen colonies of bacteria in streptomycin containing plate as well as kanamycin containing
plate, but the plate having streptomycin and kanamycin both together showed growth of only
bacterial culture which had undergone transduction whereas both culture 1 and 2 could not grow on
this plate.

Conclusion:
This experiment suggests that there is a horizontal transfer of genetic material this reason in
petriplates with streptomycin and kanamycin showed growth of E. coli which has undergone
transduction.

Precaution:
1. Work under sterile condition.
2. Discard waste as microbial waste.
3. Add antibiotic in the agar when the temperature of molten agar is ambient.
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Genetic Engineering Lab

EXPERIMENT-10
Aim: To perform transfer of gene from one bacterium to another.

Why We Need To Study Bacterial Conjugation

Genetic variability is one of the essential features of the evolution. In prokaryotic cells or haploid
cells genetic recombination takes place by 3 different processes 1) conjugation, 2) transduction and
3) transformation. Since genetic information can be passed on from one bacterial strain to another
by conjugation, therefore conjugation is helpful for gene transfer from one bacterial strain to another
bacterial strain. One of the classical examples that one can study in laboratory is transfer of antibiotic
resistance gene which occurs due to process of conjugation.

Principle
Conjugation is a mating process among bacteria in which transfer of genetic material is
unidirectional. Transfer of genetic material takes place due to physical contact between two sexually
differentiated bacterial cell types. These characteristics exist in some bacterial strains, which are
determined by the presence of “fertility factor or F factor”. The strain that lacks F factor is known
as “recipient or female” and designated as F_, while the strains which act as donor of genetic
material is known as “donor or males” and designated as F+. During mating only F_ factor is
transferred. Therefore genetic material is transferred from an F+ cell to an F_ cell via conjugation.
Genetic material through is passed through a thin tube- like structure present on F+ cell, called as F
pilus. F+ cells are referred to as having an F factor, the fertility plasmid that allows donor cell to
conjugate with the recipient via F pilus.

Steps

1. Bacterial culture
It is require growing different bacterial culture separately. Overnight grown bacteria are in log phase
or late log phase.

2. Bacterial conjugation
Mix both the bacterial strains and incubate to promote conjugation.

3. Stopping conjugation
After incubation agitate tubes to stop conjugation

4. Evaluation of conjugation
Grow bacteria on TSA agar plate in the presence of specific antibiotics to evaluate or to confirm the
process of conjugation.
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Genetic Engineering Lab

Materials
i) E. coli donor strain (chloramphenicol resistant),

ii) E. coli recipient strain (rifampicin resistant),

iii) Tryptic soy Agar medium,

iv) Bunsen burner, petri plate, Incubator, permanent marker, 70% ethanol, spreader, pipettes, etc.

Method
A. BACTERIAL CONJUGATION
1. Grow culture of F_ (E. coli recipient strain) and F+ (E. coli donor strain) overnight, so that
bacteria are in log phase.
2. Mix 0.1 ml of donor strain with 0.9 ml of recipient strain of E. coli
3. Now add 5 ml of sterile tryptic soy broth in the mixture of two different strains of bacteria.
4. Incubate tubes at 37°C for three hours or overnight.
5. Agitate tube vigorously to terminate conjugation.

B. CONFIRMATION OF CONJUGATION
1. Prepare 4 petriplates with tryptic soy agar, with the addition of following antibiotics

i) TSA
ii) TSA + chloramphenicol
iii) TSA +rifampicin
iv) TSA + rifampicin+ chloramphenicol

2. Divide petriplates into 3 sections by marking in the back and label them as F+, F_ and
conjugated.
3. Use a loop to plate the different strains of bacteria and spread them.
4. Incubate for 24 hrs. and 48 hrs. and count colonies for each strain.
5. Dispose the plates after autoclaving.
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Observations
Table 1: observations for the colonies observed after 24 hrs of incubation for bacterial conjugation.

Sample TSA TSA + TSA + TSA +

Chloramphenicol rifampicin chloramphenicol + rifampicin
F+

F_

After

Conjugation

Table 2: observations for the colonies observed after 48 hr of incubation for bacterial conjugation.

Sample TSA TSA + TSA + TSA +

Chloramphenicol rifampicin chloramphenicol + rifampicin
F+

F_

After

conjugation

RESULTS

We have seen the growth of E. coli in the presence of chloramphenicol + rifampicin added with
TSA. This suggests that F+ strain has transferred its plasmid to F_ strain of E. coli because there
were bacterial colonies in TSA plate with chloramphenicol and rifampicin resistance.