BT2307 Molecular biology Lab Manual KVCET / DBT / NE

List of Experiments in Molecular Biology Lab

S.no. Name of the Experiment Page no.

1. Agarose gel electrophoresis

2. Preparation of genomic DNA from plant tissue

3. Preparation of Genomic DNA from Bacteria

4. Preparation of Genomic DNA from Mammalian Tissue

5. Isolation of plasmid dna by alkaline lysis method

6. Restriction enzyme digestion

7. Preparation of competent cells by calcium chloride (CaCl2) method

8. Transformation

9. Plating of lambda phage

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:1
Date:
Agarose Gel Electrophoresis
Aim
To separate the given DNA sample by agarose gel electrophoresis
Principle
Agarose gel electrophoresis is a simple and highly effective method for separating,
identifying, and purifying 0.5- to 25-kb DNA fragments. The protocol can be divided into three
stages: (1) a gel is prepared with an agarose concentration appropriate for the size of DNA
fragments to be separated; (2) the DNA samples are loaded into the sample wells and the gel is
run at a voltage and for a time period that will achieve optimal separation; and (3) the gel is
stained or, if ethidium bromide has been incorporated into the gel and electrophoresis buffer,
visualized directly upon illumination with UV light.

Material required
Electrophoresis buffer (TAE)
Stock solution-50 X TAE Buffer Preparation protocol (Tris-Acetate-EDTA)

242 gm - Tris base

57.1 ml - Acetic Acid
100mL - 0.5 M EDTA (shake vigorously before use)

Add ddH2O to 1 Liter and adjust ph to 8.5 using KOH.

Working standard-1x TAE
Ethidium bromide solution
Stock solution-50 mg EtBr in 100 ml water
Working solution-0.5 microgram/ml of gel solution
Electrophoresis-grade agarose
10x loading buffer

 Weigh 25 mg bromophenol blue, 25 mg xylene cyanol FF and 4 gm Sucrose.

 Dissolve them in 9 ml deionized water.
 Adjust the volume 10 ml with deionized water

Apparatus:
 DNA molecular weight markers
 Microwave oven
 Horizontal gel electrophoresis apparatus
 Gel casting platform
 Gel combs
 DC power supply

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Procedure
1. Add 0.8gm of electrophoresis-grade agarose to 100ml of electrophoresis buffer sufficient for
constructing the gel. Melt the agarose in a microwave oven or autoclave and swirl to ensure even
mixing.
2. Seal the gel casting platform if it is open at the ends. Pour in the melted agarose and insert the
gel comb, making sure that no bubbles are trapped underneath the combs and all bubbles on the
surface of the agarose are removed before the gel sets.
3. After the gel has hardened, remove the tape from the open ends of the gel platform and
withdraw the gel comb, taking care not to tear the sample wells.
4. Place the gel casting platform containing the set gel in the electrophoresis tank. Add sufficient
electrophoresis buffer to cover the gel to a depth of about 1 mm (or just until the tops of the wells
are submerged). Make sure no air pockets are trapped within the wells.
5. DNA samples should be prepared in a volume that will not overflow the gel wells by addition
of the appropriate amount of 10xloading buffer. Samples are typically loaded into the wells with
a pipette or micropipette. Care should be taken to prevent mixing of the samples between wells.
6. Set the voltage to the desired level, typically 1 to 10 V/cm of gel, to begin electrophoresis. The
progress of the separation can be monitored by the migration of the dyes in the loading buffer.
7. Turn off the power supply when the bromophenol blue dye from the loading buffer has
migrated 3/4th of the total distance. The DNA can be visualized by placing on a UV light source
and can be photographed directly.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:2
Date:
Preparation of genomic DNA from plant tissue
AIM:
To isolate genomic DNA from the given plant tissue.

PRINCIPLE:
The isolation of high molecular weight chromosomal DNA is often the first step in molecular
cloning. The objective of this experiment is to isolate chromosomal DNA with minimum of
nicks. The resuspended cells are first mixed with extraction buffer. This buffer contains EDTA
that forms complexes with several kinds of metal ions. Divalent metal cations, such as Mg2+ are
required cofactors by the majority of enzymes. The DNA being extracted is protected from DNA
degradation since the complexed Mg2+ cannot be utilized by the enzyme. In the presence of
salts, DNA and RNA precipitate from solution containing high percentages of isopropanol or
ethanol. Smaller molecules such as sugars and amino acids remain in solution. The aqueous
phase is overlaid with ice cold isopropanol. High molecular weight DNA precipitates at the
interface of the liquids. Due to its size and abundance, chromosomal DNA forms a viscous,
clotted mass that is easily collected using a glass rod. This process is known as spooling.
Spooled DNA is redissolved in buffer and analyzed by electrophoresis.

Materials required
1. Plant tissue
2. Mortar and pestle
3. Extraction buffer
1M Tris HCl (pH-7.5) - 5ml
5M NaCl - 1.25ml
0.5M EDTA (pH-8.0) - 1.25ml
0.5% SDS -1.25ml
Bring final volume to 25ml with distilled water and autoclave.
4. Chloroform : isoamyl alcohol (24:1)
5. Ice cold isopropanol
6. 70% ethanol
7. TE Buffer
10mM Tris HCl(pH-8.0)
1mM EDTA (pH-8. 0)
Procedure
1. Collect 500 mg of experimental plant tissue and grind thoroughly in the mortar
and pestle.
2. Add 400 micro liter of prewarmed (60 degree Celsius) extraction buffer and mix
gently. Incubate at 60 degree Celsius in water bath for 30 minutes.

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

3. Add an equal volume of chloroform: isoamyl alcohol and mix well for 5 minutes
by inverting the tube several times.
4. Centrifuge at 10000 rpm for 10 minutes.
5. Carefully transfer the supernatant (Aqueous phase at the top of the viscous layer)
into a sterilized eppendorf tube.
6. Add equal volume of ice cold isopropanol .Mix gently by inversion and leave at -
20 degree Celsius for 30 minutes.
7. Centrifuge at 10000 rpm for 10 minutes and decant to drain isopropanol.
8. Invert the tube, drain on tissue paper and allow pellet to air dry. Take care that
pellets do not slip down along the tube wall.
9. Resuspend the pellet in 100 microlitre of TE buffer and store in -20 degree
Celsius.
10. Test the sample by agarose gel electrophoresis.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no: 3
Date:
Preparation of Genomic DNA from Bacteria
Aim
To isolate genomic DNA from bacteria.

Principle
Bacteria from a saturated liquid culture are lysed and proteins removed by digestion with
proteinase K. Cell wall debris, polysaccharides, and remaining proteins are removed by selective
precipitation with CTAB, and high-molecular-weight DNA is recovered from the resulting
supernatant by isopropanol precipitation.

Materials required
1. TE Buffer
10mM Tris HCl (pH-8.0)
1mM EDTA (pH-8. 0)
2. 10% sodium dodecyl sulfate (SDS)
3. 20 mg/ml proteinase K
4. 5 M NaCl
5. CTAB/NaCl solution(10% CTAB in 0.7 M NaCl)
Dissolve 4.1 g NaCl in 80 ml water and slowly add 10 g CTAB (hexadecyltrimethyl
ammonium bromide) while heating and stirring. If necessary, heat to 65°C to dissolve.
Adjust final volume to 100 ml.
6. 24:1 chloroform/isoamyl alcohol
7. 25:24:1 phenol/chloroform/isoamyl alcohol
8. Isopropanol
9. 70% ethanol

Procedure
1. Inoculate a 5-ml liquid culture with the bacterial strain of interest. Grow in conditions
appropriate for that strain (i.e., appropriate medium, drug selection, temperature) until the culture
is saturated. This may take several hours to several days, depending on the growth rate.
2. Spin 1.5 ml of the culture in a centrifuge for 2 min, or until a compact pellet forms. Discard
the supernatant.
3. Resuspend pellet in 567 µl TE buffer and add 30 µl of 10% SDS and 3 ml of 20 mg/ml
proteinase K Mix thoroughly and incubate for1 hr at 37°C.
4. Add 100 µl of 5 M NaCl and mix thoroughly.
5. Add 80 µl of CTAB/NaCl solution. Mix thoroughly and incubate for 10 min at 65°C.
6. Add an approximately equal volume (0.7 to 0.8 ml) of chloroform/isoamyl alcohol, mix
thoroughly, and spin for 5 min in a centrifuge.

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

7. Remove aqueous, supernatant to a fresh micro centrifuge tube, leaving the interface behind.
Add an equal volume of phenol/chloroform/isoamyl alcohol, extract thoroughly, and spin in a
centrifuge for 5 min.
8. Transfer the supernatant to a fresh tube. Add 0.6 volume of isopropanol to precipitate the
nucleic acids. Shake the tube back and forth until a stringy white DNA precipitate becomes
clearly visible.
9. Centrifuge the content for 5 min at room temperature.
10. Wash the DNA with 70% ethanol to remove residual CTAB and re spin for 5 min at room
temperature to re pellet it.
11. Carefully remove the supernatant and re dissolve the pellet in 100 µl of TE buffer.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:4
Date:
Preparation of Genomic DNA from Mammalian Tissue
Aim
To isolate genomic DNA from mammalian tissue

Principle
Tissue is rapidly frozen and crushed to produce readily digestible pieces. The processed tissue is
placed in a solution of proteinase K and SDS and incubated until most of the cellular protein is
degraded. The digest is deproteinized by successive phenol/chloroform/ isoamyl alcohol
extractions, recovered by ethanol precipitation, and dried and resuspended in buffer.

Materials
Tissues, whole or cultured cells
Liquid nitrogen
Digestion buffer
100 mM NaCl
10 mM Tris.Cl, pH 8
25 mM EDTA, pH 8
0.5% SDS
0.1 mg/ml proteinase K
Store at room temperature
PBS -ice cold
Na2HPO4 (anhydrous) -10.9 g
NaH2PO4 (anhydrous) -3.2 g
NaCl - 90 g
Distilled water -1000 ml
Mix to dissolve and adjust pH to 7.2 Store this solution at room temperature.
Dilute 1:10 with distilled water before use and adjust pH if necessary.
7.5 M ammonium acetate
70% and 100% ethanol
TE buffer, pH 8
Incubator or water bath at 50°C, with shaker

Procedure:
1. As soon as possible after excision, quickly mince tissue and freeze in liquid nitrogen.
2. Starting with between 200 mg and 1 g, grind tissue with a pre chilled mortar and pestle, or
crush with a hammer to a fine powder (keep the tissue fragments, if crushing is incomplete).
3. Suspend the powdered tissue in 1.2 ml digestion buffer per 100 mg tissue. There should be no
clumps.

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Beginning with tissue culture cells:

4. Pellet suspension culture out of its serum-containing medium. Trypsinize adherent cells and
collect cells from the flask. Centrifuge 5 min at 500 × g, 4°C, and discard supernatant.
5. Resuspend cells with 1 to 10 ml ice-cold PBS. Centrifuge 5 min at 500 × g and discard
supernatant. Repeat this re suspension and centrifugation step.
6. Resuspend cells in 1 volume digestion buffer. For <3 × 107 cells, use 0.3 ml digestion
buffer. For larger numbers of cells use 1 ml digestion buffer/108 cells.
7. Incubate the samples with shaking at 50°C for 12 to 18 hr in tightly capped tubes.
8. Thoroughly extract the samples with an equal volume of phenol/chloroform/isoamyl alcohol.
9. Centrifuge 10 min at 1700 × g in a swinging bucket rotor.
7. Transfer the aqueous (top) layer to a new tube and add 1⁄2 volume of 7.5 M ammonium
acetate and 2 volume (of original amount of top layer) of 100% ethanol. The DNA should
immediately form a stringy precipitate. Recover DNA by centrifugation at 1700 × g for 2 min.
8. Rinse the pellet with 70% ethanol. Decant ethanol and air dry the pellet.
9. Resuspend DNA at 1 mg/ml in TE buffer until dissolved. Shake gently at room temperature or
at 65°C for several hours to facilitate solubilization. Store indefinitely at 4°C.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:5

Date:

ISOLATION OF PLASMID DNA BY ALKALINE LYSIS METHOD

AIM

To isolate the given plasmid DNA by alkaline lysis method.

PRINCIPLE

Exposure of bacterial suspension to study anionic detergent at high pH, open the cell
wall, denatures the genomic DNA and proteins and release the plasmid DNA into the
supernatant. Although the alkaline lysis solution completely disturbs the base pairing, the strands
of close circular plasmid DNA are unable to separate from each other. As long is the intensity
and duration of exposure to hydroxides when the pH is neutral.

During lysis bacterial protein, broken cell wall and denature the genomic DNA. These
complexes are efficiently precipitated from solution. After the denatured material has been
removed by centrifugation, native plasmid DNA can be recovered from the supernatant.

MATERIALS

 LB Medium: Deionized H2O, to 950 ml

Tryptone, 10 g
Yeast extract, 5 g
NaCl, 10 g

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to
7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1 liter with deionized
H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.

 Alkaline Lysis Solution I: 50 mM glucose

25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)

Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at
15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.

 Alkaline Lysis Solution II: 0.2 N NaOH (freshly diluted from a 10 N stock)
1% (w/v) SDS

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

 Alkaline Lysis Solution III: 5 M potassium acetate, 60.0 ml

Glacial acetic acid, 11.5 ml
H2O, 28.5 ml

PROCEDURE:

PREPERATION OF CELLS:

 Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate
antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at
37°C with vigorous shaking.
 Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30
seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C.
 Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.

LYSIS OF CELL WALL:

 Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous
vortexing.
 Add 200 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension.
Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Store
the tube on ice.
 Add 150 µl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline
lysis solution III through the viscous bacterial lysate by inverting the tube several times.
Store the tube on ice for 3-5 minutes.
 Centrifuge the bacterial lysate at maxi mum speed for 5 minutes at 4°C in a microfuge.
Transfer the supernatant to a fresh tube.
 Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room
temperature. Mix the solution by vortexing and then allow the mixture to stand for 2
minutes at room temperature.
 Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes
at 4°C.
 Store the open tube at room temperature until the ethanol has evaporated and no fluid is
visible in the tube (5-10 minutes).
 Dissolve the nucleic acids in 50 µl of TE buffer (pH 8.0), containing 20 µg/ml dnase-free
Rnase A (pancreatic Rnase). Vortex the solution gently for a few seconds. Store the DNA
solution at -20°C.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:6

Date:

Restriction enzyme digestion

Aim

To digest the plasmid DNA pUC 118 and 119 by using EcoR1 and Hind III restriction
enzymes.

Principle

Restriction endo nucleases are bacterial enzymes that recognize specific nucleotide sequences
within a double stranded DNA molecule and cleave the DNA at those specific sites, referred to
as recognition sequences. The enzymes act on the double stranded DNA by cleaving the two
phosphor diester bonds one within each strand. Type – II restriction endo nucleases are used in
cloning experiments as they recognize base pair sequences 4-8 nucleotides long and cut within
these sequences, giving rise to discrete DNA fragments of defined length and sequence. Ex:
EcoR1, Hind III, XbaI. Restriction digestion has the advantage of allowing the foreign
DNA to be digested with the appropriate restriction enzymes. Alternatively, the fragment can be
inserted into the vector at any site that generates compatible termini. It can also be used in
finding the molecular weights of the different DNA fragments by running a standard marker.

Requirements:

Plasmid DNA preparations of pUC 118 and pUC 119 g

Enzymes: Eco R1 and Hind III enzymes.

Buffer: RE Buffer ‘C’.

Instruments: water bath, microfuge.

Others: Autoclaved filtered double distilled water.

METHOD OF RESTRICTION DIGESTION:

Restriction enzymes EcoR1, Hind III, 10X enzyme buffer and autoclaved filtered double distilled
water are added to the DNA samples of pUC 118 and pUC 119 g in the manner as shown in the
following table. Total reaction volume – 40µl

DNA sample DNA (µl) Buffer (µl) EcoR1 (µl) Hind III (µl) ADDW (µl)
pUC 118 30.00 6.00 0.50 0.50 3.00
pUC 119 g 30.00 6.00 0.50 0.50 3.00

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

The tubes are spun for few seconds and incubated at 370C for three hours in a water bath. The
DNA samples are analyzed directly on 0.8% agarose gel. For the purpose of comparison, the
undigested samples of pUC 118 and 119 g are also loaded. After the running, the is observed
under UV for the linearization of pUC 118 DNA vector band and pUC 119 DNA vector band
and release of the insert gene band.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:7

Date:

PREPARATION OF COMPETENT CELLS BY CALCIUM CHLORIDE (CaCl2)

METHOD

AIM:

To prepare competent cells by calcium chloride method.

PRINCIPLE:

Some species of bacteria mutually takes upon DNA at certain stage of growth called
competent. Some species of bacteria are non- competent at any stage. Competency can be
artificially induced by adding calcium chloride prior to adding DNA. The calcium destabilizes
the cell membrane and DNA is taken up during heat shock treatment. When the cells are exposed
briefly at 42°C and chilling immediately following the heat shock causes the pores in the cell
membrane to open.

MATERIALS:

 0.1 M CaCl2
 Centrifuge tubes
 LB broth
 Spectrophotometer
 Ice bucket

PROCEDURE:

 Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated
for 16-20 hours at 37°C.
 Transfer the colonies into the 100 ml of LB broth in the 1L flask.
 Incubate the culture for 3 hours at 37°C with vigorous agitation.
 Monitor the growth of the culture using a spectrophotometer.
 When the OD600 nm reaches 0.3, after the 1 ml bacterial cell to sterile eppendorf tubes.
 Cool the cultures to 0°C by storing the tubes on ice for 10 minutes.
 Recover the cells by centrifugation at 6000 rpm for 5 minutes at 4°C. Decant the medium
from the cell pellets.
 Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the
last traces of media to drain away.
 Resuspend the pellet by swirling or gentle vortexing in 1.5 ml of ice-cold 0.1 M CaCl2.

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

 Centrifuge at 6000 rpm for 8 minutes at 4°C.

 The supernatant was discarded and the pellet is resuspended in 60 µl of ice cold solution
of 0.1 m CaCl2.
 The cells are said to be competent cells which have the ability to take up the foreign
DNA. These cells can be directly used for the transformation or can be stored at 70°C up
to 48 hours.
 Aseptically aliquot 100ml of competent cells.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:8

Date:

TRANSFORMATION

AIM:

To transform the prepared competent cells by using PUC series of plasmids.

PRINCIPLE:

After the uptake of foreign DNA by competent cells and this technique is said to be
transformation. For the purpose of transformation PUC 18 which is a high copy number plasmid
containing ampicillin resistance gene along with coding information for just 146 amino acid (N-
terminal) of β galactosidase (lac Z gene being used), the host is DH5α which bases a deletion at
the amino terminal end of lac Z gene and thus, synthesis are in active C- terminal fragment on
transforming such competent bacterial strain with PUC 18 with the host and plasmid enclosed
fragments associate to form an enzymatically active protein, this type of complementation is
known as α- complementation.

MATERIALS REQUIRED:

 LB Broth
 PUC 18
 Competent cells
 Micro pipette
 Eppendorf Tube
 Water bath

PROCEDURE:

To 100 µl of competent cells, add 5 µl of plasmid DNA and gently tap the vial and keep
it in ice for 20 minutes. Heat shocks the cells by placing the vial in 42°C water bath for 2 mins.

Return the vials to chill for 5 minutes. Add 1 ml of LB broth to the vial and incubate the
culture for 1 hour at 37°C to allow the bacteria to recover and express the antibiotic. The
transformed cells are screened for the transformation efficiency. Label three LB ampicillin plates
with X-GAL and IPTG, pipette 100 µl of LB Broth on each plate. Add 50 and 150 µl of the
transformed cells in each plate respectively. Mix well and spread thoroughly using a pipette or
separator. Repeat the step for other aliquots of competent cells transformed.

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Plate the 100 µl of competent cells that has not been transformed. Label this as control
plate.

Incubate the plate at 37°C overnight.

Transformation efficiency is expressed as a number of transformant/ µg of DNA.

Result:

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

Ex.no:9

Date:

Plating of lambda phage

PRINCIPLE

This protocol describes a method for generating isolated plaques from a stock of bacteriophage .
Each plaque derives from infection of a single bacterium by a single bacteriophage particle.
Because each plaque contains the progeny of a single virus particle, the bacteriophages derived
from a single plaque are essentially genetically identical to one another.

METHOD

1. Inoculate rich medium (LB in a 250-ml conical flask) with a single bacterial colony of
the appropriate E. coli strain. Grow the culture overnight at 37°C with moderate
agitation.
2. Centrifuge the cells at 4000g (5800 rpm in a Sorvall SS-34 rotor) for 10 minutes at room
temperature.
3. Discard the supernatant, and resuspend the cell pellet in 20 ml of 10 mM MgSO4.
Measure the OD600 of a 1/100 dilution of the resuspended cells and dilute the cells to a
final concentration of 2.0 OD600 with 10 mM MgSO4.
4. Store the suspension of plating bacteria at 4°C.
5. Melt top agar or agarose by heating it in a microwave oven for a short period of time.
Store aliquots of the melted agar or agarose (3 ml for 100-mm plates, 7 ml for 200-mm
plates) on a heating block or in a water bath at 47°C to keep the solution molten.
6. Prepare tenfold serial dilution of the bacteriophage stocks (in SM plus gelatin). Mix each
dilution by gentle vortexing or by tapping on the side of the tube.
7. Dispense 0.1 ml of plating bacteria from Step 4 into a series of sterile tubes (13 or 17 x
100 mm).
8. Add 0.1 ml of each dilution of bacteriophage stock to a tube of plating bacteria. Mix the
bacteria and bacteriophages by shaking or gently vortexing.
9. Incubate the mixture for 20 minutes at 37°C to allow the bacteriophage particles to
adsorb to the bacteria. Remove the tubes from the water bath and allow them to cool to
room temperature.
10. Add an aliquot of molten agar or agarose to the first tube. Mix the contents of the tube by
gentle tapping or vortexing for five seconds and, without delay, pour the entire contents
of the tube onto the center of a labeled agar plate. Try to avoid creating air bubbles. Swirl
the plate gently to ensure an even distribution of bacteria and top agarose. Repeat the

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BT2307 Molecular biology Lab Manual KVCET / DBT / NE

procedure until the contents of all the tubes have been transferred to separate labeled
plates.
11. Replace the lids on the plates. Allow the top agar/agarose to harden by standing the plates
for 5 minutes at room temperature. Invert the closed plates and incubate them at 37°C.
Continue incubating the plates overnight, then count or select (pick) individual plaques.

Result:

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