SAVEETHA SCHOOL OF ENGINEERING

SAVEETHA INSTITUTE OF MEDICAL AND TECHNICAL

SCIENCES INSTITUTE OF BIOINFORMATICS

Course Code: BIA0819

Course Name: RECOMBINANT DNA TECHNOLOGY FOR GMOs
Course Faculty: Ms. SWETHA SHREE R

LAB MANUAL

S.No. EXPERIMENT NAME TYPE

1. SOLUTION PREPARATION- CALCULATION FOR Minor
SOLUTION I, II, III PREPARATION
AND AMPICILLIN STOCK.
2. ISOLATION OF BACTERIAL GENOMIC DNA Major

3. PLASMID DNA ISOLATION USING ALKALINE Major

LYSIS
4. RESTRICTION ENZYME DIGESTION OF PUC18 DNA Major
5. LIGATION OF DNA FRAGMENT WITH CLONING Major
VECTOR
6. PREPARATION OF COMPETENT CELLS Major
7. POLYMERASE CHAIN REACTION Major
8. AGAROSE GEL ELECTROPHORESIS Major
9. TRANSFORMATION IN E. COLI WITH Major
RECOMBINANT VECTOR BY CALCIUM CHLORIDE
METHOD
10. PRIMER DESIGNING Minor
11. NANO DROP Major
12. PREPARATION OF LB AGAR AND LB BROTH Minor
13. DESIGNING CLONING STRATEGIES Minor
14. BLUE WHITE SELECTION Major
15. ANTIBIOTIC RESISTIVITY – GRAM POSITIVE AND Minor
NEGATIVE ORGANISM
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SCIENCES INSTITUTE OF BIOINFORMATICS
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SAVEETHA INSTITUTE OF MEDICAL AND TECHNICAL
SCIENCES INSTITUTE OF BIOINFORMATICS

EXPERIMENT 1
SOLUTION PREPARATION- CALCULATION FOR SOLUTION I,II,III
PREPARATION AND AMPICILIN STOCK.
SOLUTION I PREPARATION
PRINCIPLE
Solution I is used to resuspend cells after harvesting and is usually a Tris-EDTA buffer with
glucose.
MATERIALS REQUIRED
- 50 mM Glucose
- 25 mM Tris-HCl (pH 8.0)
- 10 mM EDTA (pH 8.0)
PROCEDURE
1. Prepare the components in the following quantities for 100 mL of Solution I:
- 0.9 g Glucose
- 0.3 g Tris-HCl
- 0.372 g EDTA (disodium salt)
2. Add the glucose, Tris-HCl, and EDTA to 90 mL of distilled water.
3. Adjust the pH to 8.0 with NaOH if needed.
4. Make up the volume to 100 mL with distilled water.
5. Sterilize the solution by autoclaving or filter sterilization.
SOLUTION II PREPARATION
PRINCIPLE
Solution II is a lysis solution, used to disrupt the cell membrane and solubilize DNA.
MATERIALS REQUIRED
- 0.2 M NaOH
- 1% SDS (Sodium Dodecyl Sulfate)
PROCEDURE
1. Prepare the components in the following quantities for 100 mL of Solution II:
- 0.8 g NaOH
- 1 g SDS
2. Dissolve NaOH and SDS in 80 mL of distilled water.
3. Mix well until the SDS is fully dissolved.
4. Make up the volume to 100 mL with distilled water.
5. This solution should be prepared fresh before use and should not be autoclaved.
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SOLUTION III PREPARATION

PRINCIPLE
Solution III is a neutralization solution, used to precipitate cell debris and genomic DNA.
MATERIALS REQUIRED
- 3 M Potassium Acetate, pH 5.5
PROCEDURE:
1. Prepare the components in the following quantities for 100 mL of Solution III:
- 29.4 g Potassium Acetate
- Glacial acetic acid for pH adjustment
2. Dissolve the potassium acetate in 80 mL of distilled water.
3. Adjust the pH to 5.5 using glacial acetic acid.
4. Make up the volume to 100 mL with distilled water.
5. Sterilize by autoclaving and store at room temperature.
AMPICILLIN STOCK PREPARATION
PRINCIPLE
Ampicillin is an antibiotic used to select for bacteria that contain a plasmid with the
ampicillin resistance gene.
Concentration: 100 mg/mL (final stock concentration)
PROCEDURE:
1. Weigh 1 g of ampicillin powder.
2. Dissolve in 10 mL of sterile distilled water to achieve a concentration of 100 mg/mL.
3. Filter sterilize the solution using a 0.22 µm filter to avoid heat inactivation.
4. Aliquot into smaller tubes (e.g., 1 mL per tube) and store at -20°C.
5. Use at a final concentration of 50–100 µg/mL in LB media for selecting transformed cells.
RESULT
The above prepared solutions are essential for the plasmid isolation process, especially in
recombinant DNA technology applications, and should be prepared with care to ensure
accurate results in downstream experiments.
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EXPERIMENT 2
ISOLATION OF BACTERIAL GENOMIC DNA
AIM
To isolate the genomic DNA from E. coli cells.
PRINCIPLE:
In prokaryotes, Deoxyribose nucleic acid (DNA) is double stranded and circular, and is found
throughout the cytoplasm. The cell membranes must be disrupted in order to release the DNA
in the extraction buffer. SDS is used to disrupt the cell membrane. Nucleases apparently present
on human fingertips are notorious for causing spurious degradation of nucleic acids during
purification. DNA can be protected from endogenous nucleases by chelating Mg2++ ions using
EDTA. Mg2++ ion is considered as a necessary cofactor for most nucleases. Proteinase enzyme
is used to degrade the proteins in the disrupted cell soup. Phenol chloroform is used to denature
and separate protein from DNA. Chloroform is also a protein denaturant, which stabilizes the
rather unstable boundary between an aqueous phase and pure phenol layer. The denatured
protein forms a layer at the interface between the aqueous and the organic phases which are
removed by centrifugation. DNA released from disrupted cells is precipitated by cold absolute
ethanol or isopropanol.
MATERIALS REQUIRED:
LB Broth
E. coli DH5α cells
Reagents
TE buffer(pH 8.0)
10% SDS
Proteinase K
Phenol-chloroform mixture
5M Sodium Acetate(pH 5.2)
Isopropanol
70% ethanol
Autoclaved Distilled Water
Eppendorf tubes 2 ml
Micropipette
Microtips
Microfuge
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PREPARATION OF REAGENTS:
1. TE Buffer (pH 8.0): 10 mm Tris HCl (pH 8.0) 1 mm EDTA (pH 8.0)
2. 10% SDS: Dissolve 10 g of SDS in autoclaved distilled water.
3. Proteinase K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water.
4. Phenol-Chloroform Mixture: Mix equal volume of phenol with chloroform. Keep the
mixture on ice and add 20 ml TE buffer, extract by shaking for 15 minutes. Remove the dust
on the surface layer using a pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it
on ice. 5. 5M Sodium acetate: Dissolve 41 g of sodium acetate in 100 ml distilled water and
adjust pH with dilute acetic acid. 6. Isopropanol 7. 70% Ethanol
PROCEDURE
 2 ml overnight culture was taken and the cells were harvested by centrifugation for 10
minutes 875 µl of TE buffer was added to the cell pellet and the cells were dissolved in
the buffer by gentle mixing.
 100 µl of 10% SDS and 5µl of Proteinase K were added to the cells.
 The above mixture was mixed well and incubated at 37º C for an hour in an incubator.
 1ml of phenol-chloroform mixture was added to the contents, mixed well by inverting
and were incubated at room temperature for 5 minutes.
 The contents were centrifuged at 10000 rpm for 10 minutes at 4ºC.
 The highly viscous jelly like supernatant was collected using cut tips and was
transferred to a fresh tube.
 The process was repeated once again with phenol-chloroform mixture and the
supernatant was collected in a fresh tube.
 100 µl of 5M sodium acetate was added to the contents and was mixed gently. 2 ml of
isopropanol was added and mixed gently by inversion till white strands of DNA
precipitated out.
 The contents were centrifuged at 5000 rpm for 10 minutes.
 The supernatant was removed and 1ml 70% ethanol was added.
 The above contents were centrifuged at 5000 rpm for 10 minutes.
 After air drying for 5 minutes 200 µl of TE buffer or distilled water was added.
 10 µl of DNA sample was taken and was diluted to 2 ml with distilled water.
 The concentration of DNA was determined using a spectrophotometer at 260/280 nm.
 The remaining samples were stored for further experiments.
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PRECAUTIONS:
 Cut tips should be used so that the DNA is not subjected to mechanical disruption.
 Depending on the source of DNA the incubation period of Proteinase K should
extended.
 The phenol chloroform extraction should be repeated depending on the source of DNA
to obtain pure DNA.
 DNase free plastic wares and reagents should be used.

OBSERVATION
INTERPRETATION
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EXPERIMENT 3
PLASMID DNA ISOLATION USING ALKALINE LYSIS
AIM:
To isolate plasmid DNA from given bacterial cells using Alkaline lysis methods.
PRINCIPLE
A procedure for extracting plasmid DNA from bacterial cells is described. The method is
simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet
yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The
principle of the method is selective alkaline denaturation of high molecular weight
chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate
pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA
renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small
plasmid DNAs have been extracted by this method.
MATERIALS REQUIRED
Alkaline lysis solution I: 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0),
deion water
Alkaline lysis solution II : 0.2 N NaOH, 1% (w/v) SDS, de-ion water
Alkaline lysis solution III : 5 M potassium acetate, glacial acetic acid, de-ion water
Ethanol 70% (v/v)
Isopropanol TE-RNAase pH 8.0
PROCEDURE
1. Pour overnight grown culture to 1.5 mL labelled falcon tube.
2. Centrifugate at 14.000 rpm for 1 min.
3. Remove the supernatant from the tube.
4. Repeat step 1-3, until leaves bacterial pellet as dry as possible.
5. Add 150 µL resuspension buffer, resuspend the bacterial pellet properly by vortexing.
6. Add 200 µL lysis solution to bacterial suspension (freshly made), close the tube tightly and
mix contents thoroughly by inverting the tube 4-6 times until the solution becomes viscious.
7. Add 300 µL neutralization solution and mix contents thoroughly by inverting the tube 4-6
times.
8. Centrigufe at 14.000 rpm for 5 min. 9. Take the supernatant and transfer to a new 1.5 mL
falcon max 300 µL.
10. Add equal volume of isopropanol in the supernatant (300 µL) and mix it by inverting the
tube couple of times.
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11. Incubate in -80°C for 30 min.

12. Centrifuge at 14.000 for 5 min. 13. Remove the supernatant and add 600 µL EtOH 70%.
14. Centrifuge at 14.000 for 5 min. 15. Remove the supernatant and dry the pellet for 10-30
min.
16. Dissolve the pellet in 20-50 µL TE-RNAase pH 8.0. Confirm the plasmid with 5 µL DNA
solvent by Agarose Electrophoresis.

OBSERVATION

RESULTS
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EXPERIMENT 04
RESTRICTION ENZYME DIGESTION OF PUC18 DNA
AIM
To digest the pUC18 DNA with BamH1 enzyme
PRINCIPLE
Restriction endonucleases are the class of enzymes that are used to cleave DNA at specific sites
called Restriction sites. Every restriction enzyme has a specific restriction site at which it cuts
a DNA molecule. For example restriction sequence for BamHI is GGATCC. The most
abundantly used restriction enzymes are type II restriction enzyme which cleaves at specific
restriction site only. These endonucleases function adequately at pH 7.4 but different enzymes
vary in their requirements for ionic strength usually provided by sodium chloride (NaCl) and
magnesium (Mg²+) concentration. It is also advisable to add a reducing agent such as
dithiothreitol (DTT) which stabilizes the enzyme and prevents its inactivation. Any variation
in the concentration of NaCl or Mg can lead to changes in specificity of enzyme so that it can
cleave at additional or nonstandard restriction sequences. The restriction endonucleases
produce either sticky (specific e.g. EcoRI) or blunt ends (non-specific e.g. AluI) upon cleavage.
Also based on the number of sequences identified for cleavage they can be tetracutter (4),
hexacutter (6) or octacutter (8).

MATERIALS REQUIRED
 pUC18 DNA
 BamH1 enzyme
 10X restriction buffer
 1Kb Ladder
 Sterile water
 Agarose
 6X loading dye
 1.5 ml Sterile Vials
 Ethidium Bromide
 1X TAE buffer
PROCEDURE:
 20 µl (2.5 μg) of PUC18 DNA was taken in a fresh eppendorf.
 To this 23 µl of sterile water was added followed by the addition of 5 µl of 10X
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restriction buffer.
 Finally 2μl of BamH1 enzyme was added and the mixture was incubated at 37˚C for 2-
3 hrs. 0.8% agarose gel was prepared upon which 1 Kb ladder DNA, undigested pUC18
DNA and digested PUC18 DNA were run.
 The gel was run at 100 V for 1 hr. The results were visualized and compared in UV
illuminator.
REACTION PROTOCOL:
PUC18 DNA :
20 µl (2.5 ug) Sterile water : 23 µl 10X restriction buffer : 5 µl BamH1 : 2 µl (2 units) ---------
--
Total : 50 µl ----------- Incubate at 37˚ C for 2-3 hours.
OBSERVATION:
INTERPRETATION:
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EXPERIMENT 5
LIGATION OF DNA FRAGMENT WITH CLONING VECTOR
AIM
To perform the ligation of linearized vector with DNA fragment using T4 DNA ligase.
PRINCIPLE
The enzyme that joins the DNA fragments is called DNA ligases. The DNA ligase seals the
nicks in DNA by formation of phosphodiester bond between adjacent 3’ hydroxyl and 5’
phosphate termini. The enzyme extensively used in joining DNA fragments is T4 DNA ligase.
The Ligase joins both cohesive end as well as blunt ended DNA. The DNA fragment has poly
‘A’ at 3’ end so that it can complementally bind to the ‘T’ at the 5’ end of the vector.
MATERIALS REQUIRED
Restriction digested pUC18 vector and DNA fragment T4 DNA ligase Ligation buffer
Nuclease free distilled water (autoclaved) Agarose Gel loading dye Ethidium Bromide
Micropipettes Micro tips Microfuge 50x TAE buffer Electrophoresis unit and power supply
Microwave oven/heater UV transilluminator
PROCEDURE
Three separate vials were taken and were labelled as reaction, +ve control and –ve control. 0.5
µl (10 ng) of PCR product of DNA fragment was added to reaction and –ve control vials only.
5 µl of 2X Ligation buffer was added to each of the three vials. 1 µl (50 ng) of T- vector was
added to all the three vials respectively. 1 μl of T4 DNA Ligase (1 U) was added to reaction
and +ve control vials only. 2.5 μl, 3 μl, 3.5 μl of water was added to reaction, +ve control and
–ve control vials respectively. The total volume in each of the vials was 10 μl. The prepared
mixtures were used for transformation in bacterial cells

Materials Reaction +ve control -ve control

PCR product or 0.5μl (10 _ 0.5μl

eluted DNA ng)
fragments
10XLigation buffer 5μl 5μl 5μl
pUC18 vector 1μl (50 ng) 1μl (50 ng) 1μl (50 ng)
T4 DNA Ligase 1μl 1μl _

Water 2.5μl 3μl 3.5μl

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Total 10μl 10μl 10μl

OBSERVATION:
INTERPRETATION:
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EXPERIMENT 6
PREPARATION OF COMPETENT CELLS
AIM
To prepare bacterial competent cells for transformation.
PRINCIPLE: Competence can be artificially induced in cells by treating the cells with calcium
chloride prior to adding DNA. The calcium destabilizes the membrane and adheres to the cell
surface favoring the formation of pores for entry of DNA. The DNA is taken during the heat
shock step when the cells are exposed briefly to a temperature of 42 ˚C. Immediate chilling on
ice ensures closure of pores. Selection for cells contaminating transformed DNA is enhanced
by selection markers carried by DNA. pUC series and pBR322 have ampicillin resistant factor
which enables only the transformed cells to grow on Luria Bertaini-ampicillin plates. The pUC
plasmid also has the genes for β-galactosidase enzyme. The lacZ is a gene that has series of
unique restriction sites engineered into it such that the plasmid be cut within lacZ gene. If the
plasmids are transferred into lacZ negative strain of E. coli, they will make them lacZ positive.
If either host or plasmid encoded fragments are themselves active, they can associate to form
an enzymatically active protein. This type of complementation is known as α-
complementation. The lacZ positive bacteria, result from α-complementation and can produce
active β-galactosidase enzyme which hydrolyses X-gal (5- bromo,4-chloro,3-indolyl β-D-
galactosidase) into blue coloured compound and thereby appear as blue coloured colonies in
presence of X-gal and IPTG. Any plasmid which has been inserted with the DNA fragment in
lacZ gene will not have a functional lacZ gene and thus will produce white colonies which are
unable to cleave X-gal.
MATERIALS REQUIRED
• E. Coli DH5α cells
• 10mM CaCl2
• Glycerol 50%
• LB media 1g Tryptone 0.5 g Yeast extract 0.5 g NaCl Adjust to pH 7 with NaOH
• Ice
PROCEDURE:
1. 5 ml overnight culture of E.coli (DH5α strain) cells was grown in LB media.
In the morning, this culture was put back into 50 ml of fresh LB media in a 250 ml conical
flask.
2. It was aimed at diluting this overnight culture by at least 1/100.
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The diluted culture was grown to an OD600 of 0.2 - 0.5. (a very small pellet when cells were
grown 25ml to OD600 0.2).
3. 2 ml of the broth was taken in an eppendrof tube and centrifuged at 6,000rpm for 10min at
4oC.
4. The supernatant were discarded and the pellet was suspended in 1ml of 10mM CaCl2. The
tubes were kept in ice for 20min.
5. It was centrifuged at 6,000rpm for 10min at 4oC.
6. Supernatant was discarded and the pellet was gently suspended in 100μl of 10mM CaCl2
and 16μl of 40% glycerol.
7. The competent cells prepared were stored at 4oC. All subsequent steps should be carried
out at 4oC and the cells should be kept on ice wherever possible
OBSERVATION:
INTERPRETATION:
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EXPERIMENT 07
POLYMERASE CHAIN REACTION
AIM
To amplify the 16s DNA sequence from an isolated bacterial sample.
PRINCIPLE
PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from
deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds
nucleotides to the 3’ end of a custom-designed oligonucleotide when it is annealed to a longer
template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template
that contains a region complementary to the oligonucleotide, DNA polymerase can use the
oligonucleotide as a primer and elongate its 3’ end to generate an extended region of double
stranded DNA.
MATERIALS
Forward and reverse primers, dNTPs, gDNA template, Thermal cycler, Taq polymerase, Sterile
water, PCR reaction buffer, Micropipette, PCR tubes, Spinner.
PRIMER DESIGNING
The primers in 16s rRNA are
Forward primer - 27F
[5’-AGAGTTTGATCCTGGCTCAG-3’]
Reverse primer - 1492R
[5’-TACGGYTACCTTGTTACGACTT-3]
PRIMER PREPARATION
Take 10µm from the stock 100µm primers with 200µL TE buffer and dissolve it with 90µL of
sterile water to get volume upto 100µL. This should be done for both the primers.
dNTPs PREPARATION:
From the stock of 100µm of dNTPs, take 2.5µM of each dNTPs which are added to make 10µL
and mix it with 90µL of sterile water for 100µL.
PROCEDURE
Place thin-walled PCR tubes on ice
1. Set up a 50 μL reaction (Keep all your reagents on ice):
● 1 μL Template DNA (10 ng-500 ng)
● 5 μl 10X Taq buffer with MgCl2
● 1 μl dNTP mix (10 mM each nt)
● 1 μL Forward Primer (10 μM stock)
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● 1 μL Reverse Primer (10 μM stock)

● 0.5 μL Taq DNA Polymerase (5 units/μL)
● 40.5 μL Sterile dH2O (variable)
2. Place reaction tubes in a PCR machine.
3. Set annealing temperature 5°C below the primer melting temperature (Tm).
4. Set extension step at 1-2 minutes per kilobase of product depending on whether you
are using a polymerase with proofreading capabilities.
Note: See manufacturer’s instructions for specific instructions about extension time and
temperatures.
5. Initial Denaturation for 2 minutes at 94°C.
6. Denature for 30 seconds at 94°C.
7. Anneal primers for 30 seconds at 55°C (or 5°C below Tm).
8. Extend DNA for 2 minutes at 72°C.
9. Repeat steps 8-10 for 25-30 cycles.
10. Final Extension for 5 minutes at 72°C.
11. Run 1 μL on a gel to check size and concentration of PCR product.
MASTER MIX
1. Multiply the volume of each reagent by the number of individual PCRs you wish to
perform and add 10% extra to account for pipetting error. In this example, we have 7
different reactions (7 unique primer pairs), so we multiply each volume by 7.
2. In a single 1.5mL tube combine the following:
*10X Taq buffer with MgCl2: 5 μl x 3 reactions = 15μl total
*dNTP mix (10 mM each nt): 1µl x 3 reactions = 3µl total
*Template DNA: 1µl x 3 reactions = 3 µl total
*Sterile dH2O: 40.5 µl x 3 reactions = 121.5 µl total
*Taq DNA polymerase: 0.5 µl x 3 reactions = 1.5 µl total
3. Mix the contents by gently pipetting up and down several times. Keep the tube on
ice.
4. Add the forward and reverse primers to the thinned walled PCR tubes.
5. Add the master mix to the thin walled PCR tubes. Put 50µl – 1 µl (fwd primer) – 1
µl (rev primer) = 48 µl volume of master mix to add to each PCR tube.
6. Secure the tops to the PCR tubes, gently tap each tube to bring all the liquid to the
bottom before placing it in the PCR machine.
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OBSERVATION:
The PCR products are verified under gel electrophoresis.
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EXPERIMENT 08
Transformation in E. coli with recombinant vector By Calcium Chloride Method
AIM
To prepare competent cells using E.coli Dh5-alpha strain
PRINCIPLE
As DNA is a highly hydrophilic molecule, normally it cannot pass through the cell membrane
of bacteria. Hence, in order to make bacteria capable of internalizing the genetic material, they
must be made competent to take up the DNA. This can be achieved by making small holes in
bacterial cells by suspending them in a solution containing a high concentration of calcium.
Extra-chromosomal DNA will be forced to enter the cell by incubating the competent cells and
the DNA together on ice followed by a brief heat shock that causes the bacteria to take up the
DNA. Bacteria no longer become stable when they possess holes on the cell membrane and
may die easily. Additionally, a poorly performed procedure may lead to not enough
competence cells to take up DNA. It has been reported that a naked DNA molecule is bound
to the lipopolysaccharide(LPS) receptor molecules on the competent cell surface. The divalent
cations generate coordination complexes with the negatively charged DNA molecules and LPS.
DNA, being a larger molecule, cannot itself cross the cell membrane to enter into the cytosol.
The heat shock step strongly depolarizes the cell membrane of CaCl2-treated cells. Thus, the
decrease in membrane potential lowers the negativity of the cell’s inside potential which
ultimately allows the movement of negatively charged DNA into the cell’s interior. The
subsequent cold shock again raises the membrane potential to its original value.

Competent cells are ready to use bacterial cells that possess more easily altered cell walls by
which foreign DNA can be passed through easily. Most types of cells cannot take up DNA
efficiently unless they have been exposed to special chemical or electrical treatments to make
them competent. The standard method for making the bacteria permeable to DNA involves
treatment with calcium ions. Brief exposure of cells to an electric field also allows the bacteria
to take up DNA and this process is called as electroporation. However, some types of bacteria
are naturally transformable, which means they can take up DNA from their environment
without requiring special treatment. The exact mechanisms involved in artificial competence
are not yet known well. ln CaCl2 method, the competency can be obtained by creating pores
in bacterial cells by suspending them in a solution containing a high concentration of calcium.
DNA can then be forced into the host cell by heat shock treatment at 42oC for the process of
transformation.
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Competence is distinguished into natural competence, a genetically specified ability of bacteria

that is thought to occur under natural conditions as well as in the laboratory and induced or
artificial competence, arising when cells in laboratory cultures are treated to make them
transiently permeable to DNA.
MATERIALS REQUIRED
1.LB Broth
2.E.coli starter culture
3. 100mM Calcium Chloride
4. Microcentrifuge tubes and centrifuge tubes, Pipettes
5. 30% Glycerol.
METHOD:
1. Shake E. coli at 37 °C overnight in 3 ml of LB.
2. Add 0.5 ml of the overnight culture into 50 ml of LB in a 200-ml flask. The broth should be
prewarmed to 37 °C. Shake the culture at 37 °C.
3. Monitor OD600. When OD600 of 0.35-0.4 is reached, chill the culture on ice. When highest
transformation efficiency is not required, I simply harvest cells 100-120 minutes after the
inoculation without monitoring OD600.
4. Transfer the culture to a sterile centrifuge tube, and collect cells by centrifugation at 6,000
rpm for 8 minutes at 4 °C. Discard the supernatant.
5. Resuspend the cells in 20 ml of ice-cold 50 mM CaCl2. Incubate the resuspended cells on ice
for 20 minutes. Collect cells by centrifugation as in step 4.
6. Resuspend the cells in 2.5 ml of ice-cold 50 mM CaCl2, or, if you store the competent cells
for long period as frozen stock, resuspend the cells in 2.5 ml of ice-cold 50 mM
CaCl2 containing 10% glycerol.
7. Use 100-200 microliters of the competent cells for transformation, or dispense the competent
cells into aliquotes of 100-200 microliters and freeze in liquid nitrogen for later use. This
method can be easily scaled up and down. When I want to transform an E. coli strain
quickly, I inoculate 0.05 ml of an overnight culture into 3 ml of LB, and, after shaking 100-
120 min at 37 C, cells are washed with an ice-cold calcium solution. The cells resuspended
in 100-150 microliters of the calcium solution are used for transformation.
TRANSFORMATION
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1. Thaw the competent cells on ice if they are stored frozen. Add to 100 microliters of the
competent cells appropriate amount of plasmid solutions in TE. The plasmid solution should
be less than 5 microliters.
2. Keep the cells on ice for 30 minutes.
3. Quickly transfer the tube to 42 °C water bath. Incubate for 1 minute, then transfer onto ice.
4. Add 1 ml of LB. Incubate at 37 °C for 30 minutes. Spread the cells on agar plate(s) containing
appropriate antibiotics.
OBSERVATION
RESULTS
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EXPERIMENT 09
AGAROSE GEL ELECTROPHORESIS
AIM
To determine the size of biomolecules by using the technique agarose gel electrophoresis.
PRINCIPLE
Agarose gel electrophoresis is an easy and efficient method to separate, identify and purify the
DNA molecules based on their molecular weights. In this, the negatively charged DNA
molecules migrate towards the positive charge under the influence of constant current, thus the
separation depends on the mass and charge of the DNA molecule. The agarose gel consists of
microscopic pores that act as a molecular sieve that separates molecules based upon the charge,
size and shape. Agarose gel electrophoresis can also be used to separate other charged
biomolecules such as RNA and proteins.
MATERIALS REQUIRED
Agarose powder, TBE buffer, Ethidium bromide, Blue dye, Gel caster, Electrophoresis
chamber, Power source, Gel tray, Gel comb, Electrodes, Microwave, UV transilluminator,
Pipettes, Flask, Weight balance.
BUFFER PREPARATION
1) 5X TBE for 100mL:

Buffer In grams

Tris 5.38

Boric Acid 2.75

EDTA 0.29

*Adjust the pH at 8.3

*Kept for autoclave at 121 degree celsius for at least 30 minutes
2) 1X TBE in 1000mL by sterile distilled water

Buffer In grams

Tris 10.7

Boric Acid 5.5

EDTA 0.58

EtBr STOCK PREPARATION

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*The stock was taken as 10 mg/mL and add 10g ethidium bromide solution in 1ml
*By working standard of stock could be maintained as 0.5-1µg/mL and we got 3µl
PROCEDURE:
1. A 0.8% agarose is prepared by adding 0.24g of agar in a 30 mL buffer to make a 1X
buffer.
2. It is heated for one minute in a hot oven.
3. About 3 µl of EtBr is added for staining, at hand bearable hotness.
4. The Agarose solution is poured into the boat with combs and allowed to solidify.
5. A stock base solution of 500ml with 1x TBE buffer (10ml – buffer, 490ml-distilled
water is prepared.
6. The stock solution is poured in a tray and the boat is placed in the stock solution.
7. The bands are allowed to run in the gel.
Kb 1 2 3 4

Lane1: sample with RDNA

Lane2: sample with RDNA
Lane3: sample with no DNA
Lane4: sample with DNA, RNA, PLASMID
OBSERVATION:
Bands are observed in the wells - gDNA, plasmid, RNA contamination.
RESULTS
In lane 1 and lane 2 RDNA contamination is present and in lane 3 there is no DNA, whereas
in lane 4 we can observe RDNA, DNA and PLASMID.
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EXPERIMENT 10
PRIMER DESIGNING
AIM
To design specific primers for the amplification of a target DNA sequence for use in
recombinant DNA technology applications such as gene cloning, PCR, or sequencing.
PRINCIPLE
Primer design is based on selecting short, single-stranded DNA sequences that are
complementary to the target region flanking the DNA of interest. Primers typically range from
18-25 nucleotides in length, providing specificity for the target region. Proper primer design
minimizes non-specific binding, avoids primer-dimer formation, and ensures efficient and
accurate DNA amplification.
Key factors in primer design include:
- Specificity: Primers must match the target sequence precisely.
- Length: Typically 18-25 nucleotides for optimal binding and specificity.
- Melting Temperature (Tm): The Tm of each primer should be around 55-65°C and ideally
within 2°C of each other.
- GC Content: A GC content of 40-60% provides stability.
- Avoidance of Secondary Structures:** Secondary structures like hairpins and primer
dimers reduce primer efficiency.
- 3’ End Stability: The 3' end should end with a G or C nucleotide to strengthen binding and
prevent mispriming.
PROCEDURE
1.Determine the Target DNA Sequence
- Obtain the sequence of the DNA region you wish to amplify, which can be found in genetic
databases (e.g., NCBI) or may be provided by previous sequencing data.
- Identify the region flanking the target DNA, ensuring that the primers will amplify the
sequence of interest.
2.Select Primer Parameters
- Length: 18-25 bases.
- GC Content: 40-60% for stable binding.
- Melting Temperature (Tm): 55-65°C is optimal, and the difference in Tm between forward
and reverse primers should be within 2°C.
- Annealing Temperature (Ta): Generally 2-5°C lower than the Tm, though this is often
determined empirically.
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3. Design Forward Primer

- Choose the sequence within the 5' end of the target region. The primer should match the
DNA sequence exactly.
- Check for:
- GC Content and Length: Aim for balanced GC content without runs of Gs or Cs to avoid
secondary structures.
- Tm Calculation: Calculate the Tm using the formula \( Tm = 2 \times (A+T) + 4 \times
(G+C) \).
4. Design Reverse Primer
- Select the complementary sequence at the 3' end of the target region.
- Take the reverse complement of this sequence to design the primer.
- Check for:
- GC Content and Length: Avoid sequences that might cause secondary structures.
- Tm Calculation: Match the Tm as closely as possible to the forward primer.
5. Check for Secondary Structures - Use software tools (e.g., Primer3, NCBI Primer-BLAST)
to check for potential secondary structures such as hairpins, self-dimers, or cross-dimers.
- Ensure that there are no significant self-complementary regions that could form primer
dimers.
6. Evaluate Primer Specificity
- Verify primer specificity using a tool like NCBI Primer-BLAST. This step helps confirm
that the primers are specific to the target sequence without amplifying non-target DNA
sequences.
7. Final Primer Analysis and Adjustment**
- Check primer length, Tm, GC content, and ensure both forward and reverse primers match
these parameters closely.
- Make any necessary adjustments to primer length or base composition to fine-tune the Tm
or specificity.
8. Synthesize Primers
- Once designed, primers can be synthesized through commercial providers or in-house if
facilities are available.
Calculation for Primer Melting Temperature (Tm)
For a primer sequence with 10 adenines (A), 8 thymines (T), 5 guanines (G), and 2 cytosines
(C):
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OBSERVATION
RESULT
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EXPERIMENT 11
NANO DROP
AIM
To measure the concentration and purity of nucleic acids (DNA/RNA) or protein samples using
a NanoDrop spectrophotometer.
PRINCIPLE
The NanoDrop spectrophotometer uses UV-Vis absorbance to quantify nucleic acids or
proteins by measuring the absorbance of a small-volume sample. The absorbance at specific
wavelengths (e.g., 260 nm for nucleic acids) is proportional to the concentration of the sample,
based on Beer-Lambert’s Law.
For DNA/RNA:
- 260 nm is the primary wavelength to measure nucleic acids.
- 260/280 ratio indicates protein contamination, where a ratio of ~1.8 is optimal for DNA, and
~2.0 is optimal for RNA.
- 260/230 ratio assesses contamination by organic compounds or salts, with an optimal range
of 2.0-2.2.
For Proteins:
- 280 nm is used as proteins absorb maximally at this wavelength due to aromatic amino acids
like tryptophan and tyrosine.
PROCEDURE
1. Initial Setup
- Turn on the NanoDrop spectrophotometer and select the appropriate application (e.g.,
"Nucleic Acid" for DNA/RNA, "Protein A280" for proteins).
- Ensure that the spectrophotometer software is ready for measurements and that the anoDrop
is calibrated if required.
2. Blank Measurement
- Wipe the measurement pedestal with a clean, lint-free tissue to remove any residues.
- Pipette 1-2 µL of the blank solution (e.g., nuclease-free water for DNA/RNA or the
appropriate buffer for proteins) onto the lower measurement pedestal.
- Lower the upper arm to form a liquid column between the pedestals.
- Click “Blank” on the software to zero the instrument.
- Once blanking is complete, carefully lift the arm and wipe the blank solution off both
pedestals with a clean, lint-free tissue.
3. Sample Measurement
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- Wipe the pedestal again to ensure no residues remain from the blank.
- Pipette 1-2 µL of your sample onto the lower pedestal.
- Lower the arm carefully to establish contact with the sample.
- Click “Measure” on the software.
- The software will display the absorbance spectrum, concentration, and purity ratios
(260/280 and 260/230 for DNA/RNA).
4. Record the Data
- Note the concentration and purity ratios displayed on the screen.
- Save the data in the software or record it manually.
- If running multiple samples, proceed with the next sample by repeating steps 2 and 3.
5. Cleaning and Final Steps
- After all measurements, wipe the pedestals with a clean, lint-free tissue to ensure no sample
residue remains.
- Shut down the NanoDrop spectrophotometer and software as per the instrument guidelines.
INTERPRETATION OF RESULTS
For DNA/RNA:
- A 260/280 ratio of ~1.8 indicates pure DNA, while a ratio of ~2.0 suggests pure RNA.
- A 260/230 ratio in the range of 2.0-2.2 generally indicates a lack of organic contaminants.
For Protein:
- The absorbance at 280 nm correlates with protein concentration. Some NanoDrop models
can calculate concentration directly based on a sample’s molar extinction coefficient if
known.

---
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EXPERIMENT 12
PREPARATION OF LB AGAR AND LB BROTH
AIM
To prepare LB agar and LB Broth
PROCEDURE
LB agar
1. Mix 25 grams of LB broth per liter of water in a beaker
2. Add 15 grams of agar per liter and stir until the powder dissolves
3. Transfer the mixture to an Erlenmeyer flask that's at least double the volume of the
mixture
4. Autoclave for at least 20 minutes on the liquid cycle
5. While the mixture is still liquid but cool enough to handle, pour it into plates in a
laminar flow hood
6. Let the agar set for about 30 minutes
7. Replace the lids, invert the plates, and store them in an airtight bag at 4 °C
LB broth
a. Mix 25 grams of pre-mixed broth powder, 10 grams of tryptone, 5 grams of yeast
extract, and 10 grams of NaCl with 1 liter of distilled water
b. Autoclave for 30 minutes at 121 °C
c. Let it cool before pouring into sterile test tubes with lids
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EXPERIMENT 13
DESIGNING CLONING STARTEGIES
Designing cloning strategies using computational tools involves several steps that leverage
bioinformatics resources to identify suitable cloning sites, primers, and vectors. Here’s a
general dry lab procedure for designing cloning strategies, including site instructions:
AIM
To Identify the Gene of Interest- Determine the gene you want to clone, its function, and its
biological significance.
Sequence Retrieval
Use databases like NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) to retrieve
the nucleotide sequence of the gene of interest.
- Go to NCBI GenBank.
- Search for your gene using the gene name or accession number.
- Download the sequence in FASTA format.
PROCEDURE TO SELECT A CLONING VECTOR
Identify suitable vectors (e.g., plasmids) for your cloning strategy based on the type of
expression required (e.g., protein expression, gene knockdown).
- Explore vector databases like Addgene (https://www.addgene.org/) to find vectors
compatible with your cloning method (e.g., restriction enzyme cloning, Gateway cloning).
- Review the vector map to identify multiple cloning sites (MCS) and features like
promoters and selectable markers.
Procedure to Identify Restriction Sites
-In Silico Analysis: Use software tools (e.g., SnapGene, NEBcutter, or Benchling) to
analyze the sequence of your gene and the selected vector to identify suitable restriction sites
for cloning.
- Open your sequence in the selected software.
- Use the restriction site analysis tool to visualize potential cutting sites in both your insert
and vector.
- Choose restriction enzymes that create compatible ends for ligation.
PROCEDURE TO DESIGN PRIMERS
Design primers flanking the gene of interest, including restriction sites for cloning.
- Use primer design tools like Primer3 (http://primer3.ut.ee/) or NCBI Primer-BLAST
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to generate primers.
- Set parameters for optimal melting temperature (Tm), length, and GC content.
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- Ensure primers include the restriction site sequences at the 5' end.
PROCEDURE TO CHECK FOR SECONDARY STRUCTURES
Use tools to analyze the potential for secondary structures or primer-dimer formations.
- Utilize tools like OligoAnalyzer (https://www.idtdna.com/pages/tools/oligoanalyzer) to
evaluate your designed primers for hairpins and dimers.
EVALUATE THE GENE AND VECTOR COMPATIBILITY
- Sequence Alignment: Ensure that the insert and vector sequences are compatible and that
there are no unintended mutations.
- Use BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to check for sequence similarity
and alignment.
SIMULATE CLONING
- In Silico Cloning: Use software to simulate the cloning process and visualize the final
construct.
- Tools like SnapGene or Vector NTI can help visualize the cloning procedure, including
digestion, ligation, and final construct.
OBSERVATION
RESULT
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EXPERIMENT 14
ANTIBIOTIC SENSITIVITY TEST – DISC DIFFUSION METHOD
AIM
To identify antibiotic resistance of particular sample for microbes.
PRINCIPLE
The disc diffusion method, also known as the Kirby-Bauer test, is a widely used technique for
determining the sensitivity of bacteria to antibiotics. It allows for the assessment of the
effectiveness of various antibiotics against specific bacterial strains. Here is a detailed protocol
for performing an antibiotic sensitivity test using the disc diffusion method:
MATERIALS NEEDED
Bacterial culture (test organism)
Mueller-Hinton agar plates
Sterile cotton swabs
Antibiotic discs (pre-prepared with specific antibiotics)
Sterile nutrient broth or saline
Sterile spreaders (glass or plastic)
Incubator (37°C)
Ruler or caliper
Marker or pen
Forceps or sterile tweezers
PROCEDURE
1. Preparation of Bacterial Culture
Inoculate Bacteria: Using a sterile loop, inoculate a few colonies of the target bacteria
into a sterile nutrient broth or saline solution. Incubate the culture at 37°C for 18-24 hours to
obtain a standardized bacterial suspension.
Standardize the Inoculum: Adjust the bacterial suspension to match a 0.5 McFarland standard
(approximately 1.5 x 10^8 CFU/mL) to ensure uniformity in inoculum concentration.
2. Inoculation of Agar Plates
Prepare Agar Plates: Label Mueller-Hinton agar plates with the test organism name and
date. Inoculate the Agar: Dip a sterile cotton swab into the standardized bacterial suspension.
Rotate the swab to remove excess fluid and then evenly spread the inoculum across the entire
surface of the agar plate in three directions (lawn inoculation), ensuring complete coverage.
3. Application of Antibiotic Discs
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Allow to Dry: Let the inoculated agar plates sit for a few minutes at room temperature to allow
the surface to dry slightly.
Place Antibiotic Discs: Using sterile forceps, place antibiotic discs on the surface of the agar
plate, pressing them gently to ensure contact with the medium. Space the discs at least 15-20
mm apart to avoid overlapping inhibition zones.
Control Plates: It is advisable to include control plates with known antibiotic susceptibility for
comparison.
4. Incubation
Incubate Plates: Invert the inoculated plates and incubate them at 37°C for 18-24 hours. Ensure
that plates are not stacked to allow for proper airflow.
5. Measurement of Inhibition Zones
Observe Results: After incubation, examine the plates for zones of inhibition around each
antibiotic disc. The zones are clear areas where bacterial growth has been inhibited.
Measure Zones: Using a ruler or caliper, measure the diameter of each inhibition zone (in mm)
from edge to edge. Measure the diameter perpendicular to the disc and take two measurements
for accuracy.
6. Interpretation of Results
Compare with Standards: Compare the measured inhibition zone diameters to standard
interpretive criteria provided by organizations like the Clinical and Laboratory Standards
Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing
(EUCAST). This will categorize the bacteria as sensitive, intermediate, or resistant to each
antibiotic.
OBSERVATION
RESULTS
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EXPERIMENT 15
Antibiotic sensitivity Test – Using Minimum Inhibitory Concentration (MIC) Testing
And E-test (Epsilometer Test)

Minimum Inhibitory Concentration (MIC) Testing

PRINCIPLE
The lowest concentration of an antibiotic that inhibits the visible growth of a microorganism
is determined. This can be performed using broth dilution or agar dilution methods.
Broth Dilution: Involves serial dilution of antibiotics in liquid culture medium and
inoculating with the test organism.
Agar Dilution: Involves incorporating antibiotics into agar and inoculating with the test
organism.
Advantages:
Provides quantitative data on antibiotic potency.
Can determine resistance levels more precisely.
Limitations:
More labor-intensive and time-consuming than disc diffusion.
Requires specific equipment and expertise.

E-TEST (EPSILOMETER TEST)

PRINCIPLE
A strip containing a gradient of antibiotic concentrations is placed on an agar plate inoculated
with the test organism. The MIC is determined by the intersection of the inhibition zone and
the strip.
Advantages:
Combines aspects of disc diffusion and MIC testing.
Provides quantitative results in a simpler format.
Suitable for fastidious organisms.
Limitations:
More expensive than disc diffusion.
Requires careful handling to avoid distortion of results.

OBSERVATION
RESULTS