practical manual on molecular biology and genetic engineering,recombinant DNA TECHNOLOGY

Molecular
Biology and
Genetic
Engineering
2018
A LAB MANUAL FOR BSC III YEAR BIOTECHNOLOGY
SARDAR HUSSAIN & KOMAL K.P.
GOVERNMENT SCIENCE COLLEGE, | CHITRADURGA

1Practical manual of Molecular Biology and Genetic Engineering
Sardar Hussain and K.P.Komal, GOVERNMENT SCIENCE COLLEGE, CHITRADURGA.
Contents: Page number
Experiment 1: Isolation of DNA from onion. 2-3
Experiment 2: Isolation of DNA from liver. 4-5
Experiment 3: Isolation of DNA from coconut. 6-7
Experiment 4: Isolation of DNA from spleen. 8-9
Experiment 5: Estimation of DNA by DPA method. 10-11
Experiment 6: Estimation of RNA by Orcinol method. 12-13
Experiment 7: Isolation of bacterial genomic DNA. 14-19
Experiment 8: Extraction of RNA from baker’s yeast. 20-21
Experiment 9: Isolation of Plasmid DNA. 22-25
Experiment 10: Separation of DNA by agarose gel electrophoresis. 26-29
Experiment 11. Demonstration of Restriction digestion. 30-33
Experiment 12. Demonstration of ligation. 34-37
Experiment no.13 study by charts. 38-48

Experiment 1: Isolation of DNA from onion.
Aim: To isolate DNA from onion.
Principle: In eukaryotic cell chromosomal DNA is found within nucleolus. It does not exist freely in the
nucleus but it is present in complex association with the proteins called nucleoproteins.
At low salinity nucleoproteins are insoluble were as other proteins are soluble. Further the Sodium
citrate in the form of saline sodium citrate inhibits the activity of deoxy ribonuclease which would
otherwise degrade DNA. At higher concentration of sodium chloride releases the DNA from the complex
and keeps it in the solution.
Addition of two volumes of chilled ethanol precipitates the DNA as fibrous white DNA precipitates
outs which can be collected easily by winding around a glass rod. The DNA thus collected in the rod is
dissolved in 5 ml of SSC buffer and re-precipitated with ethanol. The DNA is best stored as solution in SSC
buffer in cold.
Requirements:
1) Onion tissue
2) 2M sodium chloride
3) Saline sodium citrate (pH 7.4)
0.2M sodium citrate solution
0.14M Sodium chloride solution
4) Chilled absolute ethanol
5) Homogenizer (pestle and mortar)
6) Centrifuge and centrifuge tube
7) Spectrophotometer.
Procedure:
 Suspend about 5gm of onion and add about 5ml of S.S.C. Buffer and homogenize.
 Make up the volume to 10 ml after transferring the homogenate to a centrifuge tube and centrifuge
at 3000 RPM, for 10 minutes, discard the supernatant.
 The precipitate is homogenized with 5ml of S.S.C. buffer and centrifuge after making the volume to
10 ml with SSC buffer
 Centrifuge at 3000 RPM and discard the supernatant
 Then the sediment is suspended in about 10 ml of 2M NaCl and centrifuge at 6000 RPM for 15
minutes.
 Transfer the supernatant into a big test tube and add two to three volumes of chilled ethanol is
added gently mix by inverting the test tube and fibrous DNA is precipitated
 Collect the fibrous DNA by winding around a clean sterile bent glass tube.

Protocol /flowchart
Take 5gm of onion tissue
Homogenized with 20ml of SSC buffer
Centrifuged for 10 minutes@3000 rpm and
Discard supernatant
Precipitate is re homogenized with SSC buffer
Discard supernatant
The pellet is treated with 10 ml of 2M NaCl
Centrifuge at high 6000 rpm for 10 minutes
To the supernatant slowly add twice the volume of chilled ethanol
Fibrous DNA precipitates
Collect the fibrous DNA by glass rod and dissolved in 5ml of SSC buffer and reprecipitated with ethanol
DNA is stored as solution in SSC buffer in cold.
Result: DNA was isolated from onion.

Experiment 2: Extraction of DNA from liver.
Aim: To isolate DNA from liver.
buffer in cold.
Requirements:
1) sheep/goat liver
Procedure:
 Suspend about 5gm of liver and add about 5ml of S.S.C. Buffer and homogenize.
minutes.

Protocol /flowchart
Take 5gm of liver tissue
Discard supernatant
Discard supernatant
Result: DNA was isolated from liver.

Experiment 3: Extraction of DNA from coconut endosperm.
Aim: To isolate DNA from coconut endosperm.
buffer in cold.
Requirements:
1) Coconut endosperm
Procedure:
 Suspend about 5gm of coconut endosperm and add about 5ml of S.S.C. Buffer and homogenize.
minutes.

Protocol /flowchart
Take 5gm of coconut endosperm
Discard supernatant
Discard supernatant
Result: DNA was isolated from coconut endosperm.

Experiment 4: Extraction of DNA from spleen.
Aim: To isolate DNA from spleen.
buffer in cold.
Requirements:
1) Spleen
Procedure:
 Suspend about 5gm of spleen and add about 5ml of S.S.C. Buffer and homogenize.
minutes.

Protocol /flowchart
Take 5gm of spleen
Discard supernatant
Discard supernatant
Result: DNA was isolated from spleen.

Experiment 5: Estimation of DNA by DPA Method
Aim: To estimate the amount of DNA by DPA Method.
Principle: The principle underlying estimation of DNA using diphenylamine is the reaction of diphenylamine
with deoxyribose sugar producing blue-colored complex. The DNA sample is boiled under extremely acidic
conditions; this causes depuration of the DNA followed by dehydration of deoxyribose sugar into a highly
reactive ω-hydroxylevulinyl aldehyde. The reaction is not specific for DNA and is given by 2-deoxypentoses,
in general. The ω-hydroxylevulinyl aldehyde, under acidic conditions, reacts with diphenylamine to produce
a blue-colored complex that absorbs at 595 nm. The mechanism of reaction of deoxyribose sugar with
diphenylamine is shown in figure 6.1. As the sugar linked to only purine residues participates in the reaction,
the readout is only from 50% of the total number of nucleotides. As this holds true for both the known
standard and the given unknown sample, the concentration of the unknown sample can be directly
calculated from the standard graph.
Requirement:
 Standard DNA: 0.5 mg/ml of DNA.
 Saline citrate: 0.15 M NaCl.
0.15 M sodium citrate
 DPA reagent : 1 gm DPA in 100 ml g glacial acid and add 2.7 ml of conc H2SO4
Procedure:
 Filter out 1ml, 2ml, and 3ml of standard DNA Solution in to a series of clean and dry test tubes except
for the blank test tube.
 To each test tube add 6 ml of diphenylamine reagent
 The test tube are incubated for 10 minutes at boiling water both
 The total volume of all the test tube including the known was made up to 10 ml by adding distilled
water.
 The test tube were shaken well and absorbance was read at 600 nm
 The concentration of the DNA present is unknown sample was calculated by considering the standard
graph.
Result
 The amount of DNA present in the given sample was found to be …………………. µg/ml

 Observation and calculation:
Sl.No DNA in ml
Buffer
In ml
DPA
Reagent
Concentration
in µg
Keepinboilingwaterbathin10min
andthencoolthetubes.
OD at 540
nm
1 0.0 ml 2.0 ml
4mlineachtube
00
2 0.4 ml 1.6 ml 80
3 0.8 ml 1.2 ml 160
4 1.2 ml 0.8 ml 240
5 1.6 ml 0.4 ml 320
6 2 ml 0.0 ml 400
7 U.K UK

Experiment 6: Estimation of RNA by Orcinol Method
Aim: To estimate the amount of RNA present in the given solution.
Principle:
The acid hydrolysis of RNA releases the ribose sugar and this in the presence of strong acid undergoes
dehydration to form furfural Orcinol which then reacts with furfural in the presence of ferric chloride to give
green color. The intensity of which corresponds to the amount of RNA present in a given solution, which is
measured against a suitable blank at 660 nm using a colorimeter.
Requirement:
 Standard RNA:
Stock solution: dissolve 50 mg of RNA in 50 ML of hot 5% of Trichloro acetic acid solution.
Working solution: dissolve 10 ml of stock solution to 50 ml with 5% of TCA to get 200 µg/ml
solution
 Orcinol reagent : dissolve 1 gm Orcinol in 100 ml of 12 N HCl containing 0.5 gm of Fecl3
Procedure:
 Pipette out different aliquots of 0, 0.2, 0.4, and 0.6 …up to 1.0 ml of standard RNA solution (200
µg/ml) into different test tubes and make up the volume in each tube to 3ml by 5% TCA solution.
 5% TCA with the reagent is used as blank.
 Add 3ml of Orcinol reagent to each tube.
 Heat the test tubes in boiling water bath for 10 minutes and cool.
 The test tube were shaken well and absorbance was read at 660 nm
 The concentration of the RNA present is unknown sample was calculated by considering the standard
graph.
Result
 The amount of RNA present in the given sample was found to be …………………. µg/ml

 Observation and calculation:
Sl.No. RNA in ml
Vol.of 5%TCA
In ml
DPA
Reagent
Concentration
in µg
Keepinboilingwaterbathin10min
andthencoolthetubes.
OD at 660
nm
1 0.0 3.0
4mlineachtube
00
2 0.2 2.8 40
3 0.4 2.6 80
4 0.6 2.4 120
5 0.8 2.2 160
6 1.0 2.0 200
7 U.K U.K.

Experiment 7: Bacterial genomic DNA extraction DNA
Aim: To extract and analyze genomic DNA from bacterial cells (using spin columns).
Introduction: The term bacteria is a plural form of the Latin bacterium, meaning “staff” or “rod”. Bacteria
are amongst the most abundant prokaryotic organisms and have been on earth since almost 3.5 billion
years. They have adapted to more living conditions than any other group of organisms. They inhabit air,
soil, and water and exist in enormous numbers on surface of virtually all plants and animals. Most of the
genetic information in a bacterial (or prokaryotic) cell is contained within the chromosome, where a single
molecule of DNA is arranged as a double helix, usually in a closed loop. Escherichia coli (E. coli) are
amongst the most commonly found bacteria. They are abundant in human and animal intestine and very
easy to grow in the laboratory.
The isolation of genomic DNA from a bacterium (E. coli) generally comprises of three stages:
 Cultivation of the cells
 Disruption to release cell contents
 Purification of the DNA.
Bacterial Genomic DNA Extraction Teaching Kit (Column Based) provides a fast and easy method
for purification of total DNA for reliable applications in PCR, library screening and sequencing etc. It
is fast, simple and does not contain harmful organic compounds such as phenol and chloroform.
The DNA purification procedure using the miniprep spin columns comprises of three steps:
 Adsorption of DNA to the membrane
 Removal of residual contaminants
 Elution of pure genomic DNA
Principle: HiPer® Bacterial Genomic DNA Extraction Teaching kit simplifies isolation of DNA from bacteria
by the spin column procedure. Bacterial cells are grown in the medium till they reach log phase and are
harvested by centrifugation. After harvesting, the bacterial cell wall is degraded by Proteinase K digestion
and lysis. Following lysis, the DNA is allowed to bind to the silica-gel membrane of the HiElute Miniprep
Spin column.
HiElute Miniprep Spin Column eliminates the need for alcohol precipitation, expensive resins, and harmful
organic compounds such as phenol and chloroform, otherwise employed in traditional DNA isolation
techniques. DNA binds specifically to the advanced silica-gel membrane while contaminants pass through.

The adsorbed DNA is washed to remove trace salts and protein contaminants resulting in the elution of
high quality DNA in the Elution Buffer provided with the kit. Kit Contents: The kit can be used to extract
genomic DNA from Gram negative bacteria.
Materials Required:
Glass wares: Conical flask, Measuring cylinder, Beaker Reagents: Ethanol (96-100%), Ethidium bromide (10
mg/ml) Other requirements: UV Spectrophotometer, Tabletop micro centrifuge (with rotor for 2.0 ml
tubes), Electrophoresis apparatus, Incubator, UV Transilluminator, Micropipettes, Tips, Vortex Mixer,
Adhesive tape, Water bath or Heating block, Microwave/Burner/Hotplate.
Kit contents:
Bacterial Cell Pellets ,Lysis Solution , Lysis Solution, Prewash Solution ,Wash Solution , Elution Buffer 2.5 ml
, Proteinase K Solution ,RNase A Solution , HiEluteTM Miniprep Spin Column (in PW1139 Collection Tube) 11
Nos. Collection Tube, Polypropylene (2.0 ml) 22 Nos. Agarose 4.8 g, 50X TAE 120 ml, 6X Gel Loading Buffer.
Important Instructions:
1. Read the entire procedure carefully before starting the experiment
. 2. Thaw all refrigerated samples before use.
3. Preheat a water bath or heating block to 55oC.
4. Thoroughly mix the reagents. Examine the solutions for any kind of precipitation, if any solution (other
than enzymes) forms a precipitate warm at 55-65oC until the precipitate dissolves completely, allow it to
cool down to room temperature before use.
5. Ensure that only clean & dry eppendorf tubes and tips are used for the procedure.
Procedure:
Read the important instructions before starting the experiment.
1. Allow the bacterial cell pellet collection tube to thaw at room temperature.
2. Resuspension of cell pellet and lysis Add 180 µl of Lysis Solution I and Resuspend the pellet by gentle
pipetting.

3. Add 20 µl of Proteinase K solution to the above collection tube, vortex thoroughly for 10-15 seconds, and
incubate for 30 minutes at 55oC.
4. Add 20 µl of RNase A solution to the above collection tube, vortex thoroughly for 10-15 seconds, and
incubate for 5 minutes at room temperature (15-25°C). NOTE: This step helps in getting RNA-free genomic
DNA.
5. Add 200 µl of Lysis Solution II, vortex thoroughly for about 15 seconds, and incubate at 55oC for 10
minutes.
NOTE: A homogeneous mixture is essential for efficient lysis. 5. Prepare for binding Add 200 µl of ethanol
(95-100%) to the lysate and mix thoroughly by gentle pipetting. NOTE: A white precipitate may form on
addition of ethanol. This precipitate does not interfere with the DNA isolation procedure or with any
subsequent application. It is essential to apply all of the precipitate to the HiElute Miniprep Spin column.
6. Load lysate into the HiElute Miniprep Spin Column Transfer the entire lysate obtained from step 5 into
the HiElute Miniprep Spin column for binding the DNA. Centrifuge at 10,000 rpm for 1 minute. Discard the
flow-through liquid and place the spin column in the same 2.0 ml collection tube.
NOTE: Use a wide bore pipette tip to reduce shearing of the DNA when transferring contents onto the
column. If the solution has not completely passed through the membrane, centrifuge again at 13,000 rpm
until all the solution has passed through. Centrifugation at high speed will not affect the yield or purity of
the DNA.
7. Prewash Add 500 µl of Prewash Solution to the HiElute Miniprep Spin column and centrifuge at 10,000
rpm for 1 minute. Discard the flow-through liquid and re-use the same collection tube with the column.
8. Add 500 µl of Wash Solution to the column and centrifuge for 3 minutes at 14,000 rpm to dry the
column. Discard the flow through and place the column in the same collection tube. Centrifuge the column
for an additional 1 minute at 14,000 rpm to remove the traces of Wash Solution.
9. DNA Elution Pipette 200 µl of the Elution Buffer directly into the column without spilling to the sides.
Incubate for 1 minute at room temperature. Centrifuge at 10,000 rpm for 1 minute to elute the DNA. NOTE:
To increase the elution efficiency, incubate for 5 minutes at room temperature after adding the Elution
Buffer, then centrifuge. Elution with volumes less than 200 µl increases the final DNA concentration in the
eluate significantly, but slightly reduces the overall DNA yield. Storing DNA in water can cause acid
hydrolysis. Storage of the eluate with purified DNA: The eluate contains pure genomic DNA. For short term

storage (24- 48 hours) of the DNA, 2-8oC is recommended. For long-term storage, -20oC or lower
temperature (-80oC) is recommended. Avoid repeated freezing and thawing of the sample which may
cause denaturing of DNA. The Elution Buffer will help to stabilize the DNA at these temperatures.
Agarose Gel Electrophoresis:
Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile
distilled water*. Mix well before use. Preparation of Agarose gel: To prepare 50 ml of 0.8% agarose gel, add
0.4 g agarose to 50 ml 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot
plate, swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of
the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60 C. Add 0.5µl
Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30
minutes at room temperature. NOTE: Ethidium bromide is a powerful mutagen and is very toxic.
Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is
recommended.
Loading of the DNA samples:
To prepare sample for electrophoresis, add 2 µl of 6X gel loading buffer to 10 µl of DNA sample. Mix well by
pipetting and load the sample onto the well. Load the Control DNA after extracting the DNA sample.
Electrophoresis:
Connect the power cord to the electrophoretic power supply according to the conventions: Red-Anode
and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an
appropriate distance, depending on the size of DNA to be visualized. * Molecular biology grade water is
recommended
Quantitation of DNA:
Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the purity
of the genomic DNA. Use Elution Buffer to dilute samples and to calibrate the spectrophotometer,
measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz micro cuvette. Absorbance
readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used to correct background
absorbance. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280
nm.

Concentration of DNA sample (µg/ml) = 50 x A260 x dilution factor
Observation and Result:
Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator and calculate the
yield and purity using UV Spectrophotometer.

Calculate the concentration of isolated DNA using following formula: Concentration of DNA sample
(µg/ml) = 50 x A260 x dilution factor
Interpretation: The data in Lane 1 and 2 demonstrates that highly purified bacterial genomic DNA has been
obtained with no visible RNA contamination when electrophoresed on agarose gel. If RNA contamination
is present, one would see a faint and smeary RNA band below the genomic DNA as shown in lane 3 since
RNA being of lower molecular weight runs faster than the genomic DNA. RNA contamination is observed
when the RNase treatment has not been carried out properly. Bacterial Genomic DNA RNA contamination
9 an absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/ml of DNA. If the A260/A280 ratio is
1.6- 1.9, then the isolated DNA sample is considered to be pure. If higher A260/A280 ratio is observed it
indicates the possibility of RNA contamination.

Experiment 8: Isolation of RNA from Baker’s yeast
Aim: To isolate the total RNA from baker’s yeast (S. cerevisiae) by phenol extraction method.
Principle: Yeast total RNA is isolated by abstracting whole cell homogenate by phenol, causing denaturation
of proteins. Thus, obtained turbid suspension is centrifuged, which leads to the appearance of two phases.
The lower phenol phase containing DNA and the upper phase with carbohydrate and RNA. The RNA is then
precipitated in alcohol. The RNA product thus obtained is free of DNA but usually still contaminated with
polysaccharides. For the purification or to eliminate the polysaccharide content, it can be treated with α-
Amylase.
Materials required:
 Dry yeast- 15 g
 Phenol solution (900g/lit)
 Potassium acetate -200g/lit (pH 5.0)
 Absolute alcohol
 Diethyl ether
 Water bath(370C)
Procedure:
 Suspend the 15g of yeast in 120ml of warm water.
 After 15min about 150ml of phenol is added and the suspension is mechanically stirred for about
30min. This disrupts the protein- nucleic acid association.
 The resulting mixture is centrifuged at 3000rpm for 10min to separate the layers.
 Two layers will be seen. The lower phenol layer contains DNA along other things and the upper phase
contains RNA and along with other material like polysaccharides.
 The denatured proteins forms a thick matrix at the interphase.
 The upper aqueous phase/layer is carefully removed with the long tip pipette and filtered through a
funnel packed with glass wool.
 Prepare 20% solution of potassium acetate and adjust the pH to 5.
 Measure the volume of the filtrate and add potassium acetate solution, so that the final volume is 2%
(1ml of acetate to 9ml of filtrate).
 Add twice the volume of ethanol and leave it in cold, preferable overnight, when RNA precipitated
out.

 The powdery precipitate is collected by centrifugation, washed first with alcohol: ether mixture (3:1)
followed by ether and allowed to dry.
 The preparation will be impure as it may contain polysaccharide also.
Protocol /flowchart
Take 15gm of yeast
Suspend in 120ml of warmed water


Leave it for 15 minutes


Add 150ml of phenol and the suspension is mechanically stirred for about 30min.
Centrifuged at 3000rpm for 10min to separate the layers.
Two layers will be seen. The lower phenol layer contains DNA along other things and the upper phase
contains RNA and along with other material like polysaccharides.
The upper aqueous phase/layer is collected, filtered and filtrate is collected.
Add potassium acetate and add twice the volume of ethanol and leave it overnight.
Result: The RNA was isolated from yeast.

Experiment 9: Isolation of Plasmid DNA
Aim: To isolate the plasmid DNA by alkaline lysis method.
Principle: Plasmid are double stranded, circular, self-replicating, extra chromosomal DNA molecule. They
are commonly used as cloning vector in molecular biology. Many methods are used to isolate the plasmid
DNA. It involves three steps, they are,
Alkaline Lysis:
Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such
as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then
allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a
strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali
denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a
series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular
debris is removed and the plasmid is isolated and purified.
Sodium Dodecyl Sulfate:
Sodium dodecyl sulfate (SDS) (C12H25SO4Na) or sodium lauryl sulfate (SLS) is an anionic surfactant. It is
a molecule having a tail of 12 carbon atoms, attached to a sulfate group. This sulfate group provide the
amphiphilic properties (required for a detergent) to the molecule.
SDS has not been proven to be carcinogenic when either applied directly to skin or consumed. It has
however been shown to irritate the skin of the face with prolonged exposure of more than an hour.
Proteins are contaminating agents in any type of DNA isolation so as in plasmid DNA isolation also. They
can interfere with the final product and result with low yield. SDS is used to denature the proteins and
facilitate the DNA purification process.
Agarose gel electrophoresis is a powerful separation method frequently used to analyze plasmid DNA. The
microscopic pores present in agarose gels act as a molecular sieve. Samples of DNA can be loaded into
wells made in the gel during molding. When an electric field is applied, the DNA molecules are separated
by the pores in the gel according to their size and shape. Generally, smaller molecules pass through the
pores more easily than larger ones. Since DNA has a strong negative charge, it will migrate towards the
positive electrode in the electrophoresis apparatus. The rate at which a given DNA molecule migrates
through the gel depends not only on its size and shape, but also on the type of electrophoresis buffer, the
gel concentration and the applied voltage. Under the conditions that will be used for this experiment, the
different forms of the same plasmid DNA molecule have the following rates of migration (in decreasing
order): Super coiled > linear > Nicked Circles >dimer > trimer > etc.
Requirements for Plasmid Isolation:
Micro centrifuge.
Water bath (37°C).

Automatic micropipettes with tips.
95-100% isopropanol Ice.
Buffers and Solutions:
Alkaline lysis solution I.
Alkaline lysis solution II.
Alkaline lysis solution III.
Antibiotic for plasmid selection.
Ethanol.
Phenol: chloroform (1:1, v/v).
STE.
TE (pH 8.0) containing 20 μg/ml RNAse A.
Media: Rich medium.
Procedure:
1. 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.
2. 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.
3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
4. Resuspend the bacterial pellet in 100 μl of ice-cold alkaline lysis solution I by vigorous vortexing.
5. Add 200 μl of freshly prepared alkaline lysis solution II to each bacterial suspension. Close the tube tightly,
and mix the contents well by inverting the tube. Do not vortex! Store the tube in ice.
6. 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 in ice for 3-5 minutes.
7. Centrifuge the bacterial lysate for 5 minutes at maximum speed at 4°C in a microfuge. Collect the
supernatant to a fresh tube.
8. (Optional) Add equal volume of phenol: chloroform. Mix the organic and aqueous phases by vortexing and
then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous
upper layer to a fresh tube.
9. 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.
10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a
microfuge.
11. Discard the supernatant by aspiration. Stand the tube in an inverted position on a paper towel to allow all
of the fluid to drain away. Use a Kim wipe or disposable pipette tip to remove any drops of fluid adhering to
the walls of the tube.
12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by
centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.

13. Remove all of the supernatant by aspiration. Take care with this step, as the pellet sometimes does not
adhere tightly to the tube.
14. Remove any beads of ethanol from the tube. Store the open tube at room temperature until the ethanol
has evaporated and no fluid is visible in the tube (5-10 minutes).
15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase).
Vortex the solution gently for a few seconds and store the DNA at -20°C.
Recipes for Buffers, Solutions and Media:
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, sterilize by autoclaving and store at
4°C.
(For plasmid preparation.)
Alkaline Lysis Solution II:
0.2 N NaOH (freshly diluted from a 10 N stock).
1% (w/v) SDS.
Prepare Solution II fresh and use at room temperature.
Alkaline Lysis Solution III:
5 M potassium acetate, 60.0 ml.
Glacial acetic acid, 11.5 ml.
H2O, 28.5 ml.
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution
at 4°C and transfer it to an ice bucket just before use.
EDTA:
To prepare 0.5 M EDTA (pH 8.0): Dissolve 186.1 g of disodium EDTA•2H2O in 800 ml of Distilled 2H2O. Stir
well on a magnetic stirrer. EDTA will not dissolve into solution until the pH of the solution is reached to ~
8.0. So the pH should adjust to 8.0 with NaOH (~ 20 g of NaOH pellets) and make up the final volume to
1000ml with distilled water. Prepare the aliquots and sterilize by autoclaving.
Glycerol:
To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology grade glycerol in 9 volumes of sterile
pure H2O. Sterilize the solution by passing it through a pre rinsed 0.22-μm filter. Store in 200-ml aliquots at
4°C.
LB Media:
Deionized H2O, to 950 ml.
Tryptone, 10 g.
Yeast extract, 5 g.
NaCl, 10 g.

To prepare LB (Luria-Bertani) medium, shake the ingredients, mentioned above with Distilled water until
the solutes have dissolved. Adjust pH to 7.0 with 5 N NaOH and make up the final volume of the solution to
1 liter with deionized H2O. Then sterilize it for 20 minutes by autoclaving at 15 psi.
NaCl:
To prepare 5 M NaCl : Dissolve 292 g of NaCl in 800 ml of sterile H2O and the volume is make up to to 1
liter with deionized H2O. Prepare the aliquots and sterilize it by autoclaving.
NaOH:
To 800 ml of H2O, add 400g of NaOH pellets slowly, stirring continuously. After dissolving the pellets,
completely, make up the final volume to 1 liter with sterile H2O. Store the solution at room temperature.
Potassium Acetate:
5 M potassium acetate, 60 ml.
Glacial acetic acid, 11.5 ml.
H2O, 28.5 ml.
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution
at room temperature
SDS:
Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of SDS in 900 ml of H2O.
Heat to a temperature of 68°C and stir with a magnetic stirrer to help dissolution. Adjust the volume to 1
liter with distilled H2O. Store at room temperature. Autoclaving not necessary.
STE:
10 mM Tris-Cl (pH 8.0).
0.1 M NaCl.
1 mM EDTA (pH 8.0).
Sterilize the solution by autoclaving and store at 4°C.
TE:
100 mM Tris-Cl (desired pH).
10 mM EDTA (pH 8.0).
(10x Tris EDTA) Sterilize the buffer by autoclaving and store at room temperature.
Tris-Cl:
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH by adding concentrated HCl, to the desired
value. The volume of the solution is make up to 1 liter with distilled H2O. Prepare the aliquots and sterilize
by autoclaving.
Result: The plasmid was isolated from the bacterial sample.
Experiment 10: Separation of DNA by agarose gel electrophoresis

Aim: To separate the DNA fragments based on their Molecular weight.
Theory
Agarose gel electrophoresis is the easiest and most popular way of separating and analyzing DNA. Here
DNA molecules are separated on the basis of charge by applying an electric field to the electrophoretic
apparatus. Shorter molecules migrate more easily and move faster than longer molecules through the pores
of the gel and this process is called sieving. The gel might be used to look at the DNA in order to quantify it
or to isolate a particular band. The DNA can be visualized in the gel by the addition of ethidium bromide.
Agarose is a polysaccharide obtained from the red algae Porphyra umbilicalis. Its systematic name is (1 4)-3,
6-anhydro-a-L-galactopyranosyl-(1 3)-β-D-galactopyranan. Agarose makes an inert matrix. Most agarose
gels are made between 0.7% and 2% of agarose. A 0.7% gel will show good separation for large DNA fragments
(5-10kb) and a 2% gel will show good resolution for small fragments with size range of 0.2-1kb. Low
percentage gels are very weak (Note: - it may break when you lift them) but high percentage gels are usually
brittle and do not set evenly. The volume of agarose required for a minigel preparation is around 30-50ml
and for a larger gel, it is around 250ml.
Structure of agarose
Factors Affecting the Movement of DNA:
Voltage Applied
The migration rate of the linear DNA fragments through agarose gel is proportional to the voltage applied
to the system. As voltage increases, the speed of DNA also increases. But voltage should be limited because
it heats and finally causes the gel to melt.
Ethidium Bromide (EtBr)
It is an intercalating agent which intercalates between nucleic acid bases and allows the convenient
detection of DNA fragments in gel. When exposed to UV light, it will fluoresce with an orange colour. After
the running of DNA through an EtBr-treated gel, any band containing more than ~20 ng DNA becomes
distinctly visible under UV light. EtBr is a known "mutagen", however, safer alternatives are available. It can
be incorporated with agarose gels or DNA samples before loading, for visualization of the fragments.
Binding of Ethidium bromide to DNA alters its mass and rigidity, and thereby its mobility.
Buffers
Several different buffers have been recommended for electrophoresis of DNA. The most commonly used
buffers are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). The migration rate of DNA fragments in
both of these buffers is somewhat different due to the differences in ionic strength. These buffers provide
the ions for supporting conductivity.
Conformation of DNA

DNA with different conformations that has not been cut with a restriction enzyme will migrate with
different speeds. Nicked or open circular DNA will move slowly than linear and super coiled DNA (slowest
to fastest: nicked or open circular, linear, or super coiled plasmid). Super helical circular, nicked circular and
linear DNAs migrate gels at different rates through agarose gel. The relative nobilities of these three forms
depend on the concentration, type of agarose used to make the gel, applied voltage, buffer, and the
density of super helical twists.
Materials Required:
Buffers and Solutions:
Agarose solutions.
Ethidium bromide.
Electrophoresis buffer.
Nucleic Acids and Oligonucleotides:
DNA samples.
DNA Ladders.
(Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or
bacteriophage DNA of known sequence).
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple
and include:
 An electrophoresis chamber and power supply.
 Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.
 Sample combs, around which molten agarose is poured to form sample wells in the gel.
 Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
 Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample
wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the
electrophoresis has proceeded.
 Ethidium bromide, a fluorescent dye used for staining nucleic acids.
 Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.
NOTE: Always wear protective eyewear when observing DNA on a Transilluminator to prevent damage
to the eyes from UV light.
1. Prepare a 50x stock solution of TAE buffer in 1000m of distilled H2O:

For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.
Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water.
EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH
meter. Make the solution 100ml by adding distilled water.
Pipette out 57.1 ml of glacial acetic acid.
Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to
1000ml
2. Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the
gel:
For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding
98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA
It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the
gel preparation.
3. Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:
For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.
Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear
DNA Molecules (kb)
0.3 5-60
0.6 1-20
0.7 0.8-10
0.9 0.5-7
1.2 0.4-6
1.5 0-2-3
2.0 0.1-2
4. Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a microwave oven
until the agarose dissolves. The agarose solution can boil over very easily so keep checking it. It is good to
stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out
whereupon it boils out all over you hands. So wear gloves and hold it at arm's length. You can use a Bunsen
burner instead of a microwave - just remember to keep watching it.
5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has
cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling.
(For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O. Stir on a magnetic
stirrer for several hours to ensure that the dye has dissolved. Wrap the container in aluminum foil or transfer
the 10 mg/ml solution to a dark bottle and store at room temperature.)
6. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel.
7. Pour the warm agarose solution into the mold.
(The gel should be between 3 - 5 mm thick. Check that no air bubbles are under or between the teeth of
the comb.)

8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of
electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis
buffer. Mount the gel in the electrophoresis tank.
9. Add just enough electrophoresis buffers to cover the gel to a depth of approx. 1mm.
10. Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.
11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette or an
automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary tube. Load size standards into
slots on both the right and left sides of the gel.
12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive
anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative
electrodes). If the electrodes are 10cm apart then run the gel at 50V. It is fine to run the gel slower than this
but do not run it any faster. Above 5V/cm the agarose may heat up and begin to melt with disastrous effects
on your gel's resolution. If the leads have been attached correctly, bubbles should be generated at the anode
and cathode.
13. Run the gel until the bromophenol blue and xylenecyanol FF have migrated an appropriate distance through
the gel.
(The presence of ethidium bromide allows the gel to be examined by UV illumination at any stage
during electrophoresis).
14. The gel tray may be removed and placed directly on a transilluminator. When the UV is switched on we can
see orange bands of DNA.
CAUTION:
 Ethidium bromide is a mutagen and should be handled as a hazardous chemical (so wear gloves while
handling)
Experiment 11. Demonstration of Restriction digestion.
Aim: To perform restriction digestion of Lambda (λ) DNA using EcoRI and HindIII enzymes.

Introduction: In 1978, the Nobel Prize for Medicine was awarded to Werner Arber, Daniel Nathans and
Hamilton Smith for their discovery of restriction endonucleases, which led to the development of
recombinant DNA technologies. The first practical use of restriction enzymes in science and medicine was
the manipulation of E. coli bacteria to express recombinant human insulin for the treatment of diabetics.
The restriction enzymes have been discovered in many different bacteria and other unicellular organisms.
These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of
bases that they recognize.
Principle: Restriction Digestion involves fragmenting DNA molecules into smaller pieces with special
enzymes called Restriction Endonucleases commonly known as Restriction Enzymes (RE). Because of this
property the restriction enzymes are also known as molecular scissors. The restriction enzymes are named
from the cellular strain from which they are isolated. Restriction enzymes recognize specific sequences in
the double stranded DNA molecule (for example GATATC) and then cut the DNA to produce fragments,
called restriction fragments. The target site or sequence which the restriction enzyme recognizes is
generally from 4 to 6 base pairs and arranged in a palindromic sequence. Once it is located, the enzyme
will attach to the DNA molecule and cut each strand of the double helix. The restriction enzyme will
continue to do this along the full length of the DNA molecule which will then break into fragments. The
size of these fragments is measured in base pairs or kilo base pairs (1000 bases).
Common Restriction Enzymes:
EcoRI HindIII
5’ GAATTC 3’ 5’ AAGCTT 3’
3’ CTTAAG 5’ 3’ TTCGAA 5’
Every restriction enzyme has unique target sites for digestion. Lambda DNA has multiple restriction sites
for both EcoRI and HindIII which result into several fragments of varying sizes.
Enzyme Source Recognition
Sequence
Cut Fragment sizes (bp)
EcoRI Escherichia coli 5’ GAATTC
3’ CTTAAG
5---G AATTC--
3’
3’---CTTAA G--
5’
21226, 7421, 5804, 5643,
4878, 3530
HindIII Haemophilus
influenzae
5’ AAGCTT
3’ TTCGAA
5’---A AGCTT---3’
3’---TTCGA A---5’,
23130, 9416, 6557, 4361, 2322,
2027, 564, 125
Application of Restriction Enzymes:
 Construction of recombinant DNA molecules
 Mapping the locations of restriction sites in DNA
 Southern Blot Hybridization
 Construction of DNA Libraries
Materials Required:

Glass wares: Measuring cylinder, Beaker Reagents: Ethidium bromide (10 mg/ml) Other requirements:
Electrophoresis apparatus, UV Transilluminator, Heating block or Water Bath, Vortex Mixer,
Micropipettes, Tips, Adhesive tape, Crushed ice, Microwave/ Hotplate/ Burner
Kit Contents:
Lambda DNA ,DNA Marker, Agarose ,Restriction Enzyme: EcoRI ,HindIII ,10 X Assay Buffer for EcoRI ,
10 X Assay Buffer for HindIII ,Molecular Biology Grade Water 0.5 ml ,50X TAE 120 ml ,6X Gel Loading
Buffer Polypropylene Tubes
* Always give a quick spin before opening the vial as the liquid material may stick to the wall or to the
cap of the vial
1. Read the entire procedure carefully before starting the experiment.
2. The restriction enzymes are temperature sensitive and should always be placed on ice during the
experiment. 3. While performing the experiment place the assay buffers and restriction enzymes on
ice.
4. Use fresh tip while adding different solution to the tube. 5. While preparing the reaction mixture
the enzymes should always be added at last.
Procedure:
1. Before starting the experiment, crush ice and place the vials containing Lambda DNA, Restriction
Enzymes and Assay Buffers onto it.
2. In this experiment Lambda DNA is digested with two restriction enzymes; EcoRI and HindIII.
3. Set up the reaction mixture as follows:
Reaction 1 (EcoRI digestion)
- Lambda (λ) DNA – 5.0 µl
- 10X Assay Buffer of EcoRI – 2.5 µl
- Milli Q water –16.5 µl
- EcoRI – 1.0 µl
Total 25 µl
Reaction 2 (HindIII digestion)
- Lambda (λ) DNA – 5.0 µl
- 10X Assay Buffer of HindIII – 2.5 µl
- Milli Q water –16.5 µl
- HindIII – 1.0 µl
Total 25 µl
4. After preparing the two reaction tubes, mix the components by gentle pipetting and tapping.
5. Incubate the tubes at 37oC for 1 hour.
6. After 1 hour incubation, immediately place the vials at room temperature (15-25oC) for 10 minutes.
7. Run the samples on agarose gel as given below.

Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer add 10 ml of 50X TAE Buffer to 490 ml of
sterile distilled water*. Mix well before use.
Preparation of agarose gel: To prepare 50 ml of 1% agarose gel, measure 0.5 g agarose in a glass
beaker or flask and add 50ml 1X TAE buffer. Heat the mixture on a microwave or hot plate or burner,
swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of
the flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60oC. Add 0.5
µl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for
about 30 minutes at room temperature.
NOTE: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions
should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.
Loading of the DNA samples:
Load 3 µl of ready to use DNA Marker into the well 1.
To prepare sample for electrophoresis, add 2 µl of 6X gel loading buffer to 10 µl of DNA samples. Mix
well by pipetting and load the samples into the well.
Electrophoresis:
Connect the power cord to the electrophoretic power supply according to the conventions: Red-
Anode and Black- Cathode. Electrophorese at 100-120 V and 90 mA until dye markers have migrated
an appropriate distance, depending on the size of DNA to be visualized.
Flowchart:
Keep all the components on ice.
Prepare reaction mixture for two restriction enzymes separately.
Mix gently and incubate at 37oC for 1 hour.
Visualize the digested bands after electrophoresing on agarose gel.
Observation and Result:
Perform Agarose Gel Electrophoresis. Visualize the DNA bands using UV Transilluminator.

After running the digested samples on agarose gel, look for the digestion pattern for the two
restriction enzymes. Compare the size of each fragment with that of the DNA marker.
Interpretation: Restriction digestion patterns of lambda DNA obtained upon treatment with EcoRI
and HindIII are markedly different which demonstrates the fact that each restriction enzyme
recognizes and cleaves only a specific base sequence unique to it. The size of the fragments can be
determined by comparing with that of the DNA marker ran on the same gel.

Experiment 12. Demonstration of ligation.
Aim: To perform ligation of Lambda (λ) HindIII digest and checking of the ligation reaction through
agarose gel electrophoresis.
Introduction: Two linear DNA molecule ends (either from the same or different molecules) can be joined
together through a process called ligation. This process involves the formation of a covalent bond
between two DNA fragments (having blunt or overhanging, complementary, ‘sticky' ends) by the help of
an enzyme named as ligase. DNA ligase forms a phosphodiester bond between the 3’ hydroxyl of one
nucleotide and the 5’ phosphate of another. This process is the key player in constructing recombinant
DNA molecule.
Principle: Recombinant DNA is made possible by two important enzymes, restriction enzymes and DNA
ligase. Restriction enzymes "cut" DNA at a specific location and DNA ligase is used to "glue" two
fragments of DNA together. DNA ligation is the process through which two DNA molecule ends from the
same or different molecules are joined together. During this process a phosphodiester bond is formed
between the 3' hydroxyl of one fragment and the 5' phosphate of another. This ligation reaction is
catalyzed by a DNA ligase enzyme which ligates DNA fragments having blunt or overhanging,
complementary, ends. It is easier to ligate molecules with complementary sticky ends than blunt ends. The
commonly used DNA ligases in nucleic acid research is T4 DNA ligase and E. coli DNA ligase. E. coli DNA
ligase is more specific for cohesive ends than T4 DNA ligase but can’t be used for cloning purpose. T4 DNA
ligase is the most versatile and commonly used ligase for DNA cloning. T4 DNA ligase is approximately
60000 dalton (60 kD) protein produced by Bacteriophage T4. This ATP dependent enzyme covalently joins
blunt or compatible cohesive ends, as well as nicks in double-stranded DNA. A 5'-phosphoryl group is
required for ligation to a 3'-hydroxyl. Generally cohesive end ligation is carried out at lower temperature
(12°C to 16°C) for the maintenance of a good balance between annealing of ends and activity of the
enzyme. Blunt end ligation can be carried out at 24°C as annealing of ends is not a factor. Due to the lack of
cohesive ends blunt end ligation is more complex compared to cohesive end ligation.
A typical ligation reaction requires the following components:
• Two or more fragments of DNA that have either blunt or compatible cohesive ends
• A buffer which contains ATP
• T4 DNA ligase.

Materials Required:
Glass wares: Measuring cylinder, Beaker Reagents: Ethidium bromide (10 mg/ml) Other requirements:
Electrophoresis apparatus, UV Transilluminator, Water Bath, Micropipettes, Tips, Adhesive tape, Crushed
ice, Microwave/ Hotplate/ Burner
From the kit
Lambda DNA- HindIII digest 0.06 ml 0.24 ml -20°C, 10X Ligase Assay Buffer 0.010 ml 0.040 ml -20°C, T4 DNA
Ligase 0.008 ml 0.025 ml -20°C 4, Molecular Biology Grade Water 0.03 ml 0.120 ml R T, Agarose 3 g 10.5 g R
T, 50X TAE 60 ml 210 ml 6X Gel Loading Buffer 0.03 ml 0.1 ml 2-8°C, Polypropylene Tube (0.5 ml)
1. Read the entire experiment carefully before starting the experiment.
2. T4 DNA ligase and 10X ligase buffers are temperature sensitive and should always be placed on ice
during the experiment.
3. Thaw the ligase buffer on ice and store immediately at -20 C.
4. Use fresh tip while adding different solution to the tube. 5. While preparing the reaction mixture the
ligase should always be added at the last.

Procedure:
1. Before starting the experiment, crush ice and place the vials containing Lambda DNA-HindIII digest, 10X
ligase buffer and T4 DNA ligase onto it.
2. In this experiment Lambda DNA-HindIII digest is ligated with T4 DNA ligase.
3. Set up the reaction mixture as follows: -
Lambda (λ) DNA- HindIII digests – 4.0 µl
- 10X Ligase Assay Buffer –--------- 1.0 µl
- Milli Q water–----------------------- 4.0 µl
- T4 DNA Ligase–-------------------- 1.0 µl
Total ------------------------------------10 µl
4. After preparing the reaction tube, mix the components by gentle pipetting and tapping.
5. Incubate the tubes at 16oC water bath for 3 hours.
6. After incubation run the samples on agarose gel as given below.
Preparation of 1X TAE:
To prepare 500 ml of 1X TAE buffer add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix
well before use. Preparation of agarose gel: To prepare 50 ml of 1% agarose gel, measure 0.5 g agarose in a
glass beaker or flask and add 50 ml 1X TAE buffer. Heat the mixture on a microwave or hot plate or burner,
swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the
flask is loose to avoid buildup of pressure). Allow the solution to cool to about 55-60oC. Add 0.5 µl
Ethidium bromide (10 mg/ml), mix well and pour the gel solution into the gel tray. Allow the gel to solidify
for about 30 minutes at room temperature. NOTE: Ethidium bromide is a powerful mutagen and is very
toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile
gloves is recommended. Loading of the DNA samples: To prepare sample for electrophoresis, take 1µl of
6X gel loading buffer and 5 µl of Lambda (λ) DNA- HindIII digest in a tube, mix well by pipetting and load
the sample into the first well. Add 2 µl of 6X gel loading buffer to the ligation mix, mix well by pipetting
and load the sample into the next well. Electrophoresis: Connect the power cord to the electrophoretic
power supply according to the conventions: Red-Anode and Black- Cathode. Electrophorese at 100-120 V
and 90 mA until dye markers have migrated an appropriate distance.

After running the ligated and unligated samples on agarose gel, check the bands of both the sample and
compare the band pattern of two samples.
Interpretation:
After running the ligated and unligated samples on agarose gel, one can observe that the seven double
stranded fragments formed by digestion of lambda DNA with HindIII are ligated by T4 DNA ligase to give a
single band.

Experiment no.13 study by charts.
A) Gene cloning
To get multiple copies of a gene or other piece of DNA you must isolate, or ‘cut’, the DNA from its source
and then ‘paste’ it into a DNA vector that can replicate (or copy) itself.
The four main steps in DNA cloning are:
Step 1. The chosen piece of DNA is ‘cut’ from the source organism using restriction enzymes.
Step 2. The piece of DNA is ‘pasted’ into a vector and the ends of the DNA are joined with the vector DNA
by ligation.
Step 3. The vector is introduced into a host cell, often a bacterium or yeast, by a process
called transformation. The host cells copy the vector DNA along with their own DNA, creating multiple
copies of the inserted DNA.
Step 4. The vector DNA is isolated (or separated) from the host cells’ DNA and purified.
DNA that has been ‘cut’ and ‘pasted’ from an organism into a vector is called recombinant DNA. Because
of this, DNA cloning is also called recombinant DNA technology.
DNA cloning is used to create a large number of copies of a gene or other piece of DNA. The cloned DNA
can be used to:
 Work out the function of the gene
 Investigate a gene’s characteristics (size, expression, tissue distribution)
 Look at how mutations may affect a gene’s function
 Make large concentrations of the protein coded for by the gene

B) PCR
 Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in
a test tube rather than an organism).
 PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed
specifically for the DNA region of interest.
 In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many
copies of the target region to be produced.
The basic steps are:
Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides
single-stranded template for the next step.
Annealing (555555 - 656565°C): Cool the reaction so the primers can bind to their complementary
sequences on the single-stranded template DNA.
Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing
new strands of DNA.
PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics,
and forensic analysis of DNA.Typically, the goal of PCR is to make enough of the target DNA region that it
can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent
for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
PCR is used in many areas of biology and medicine, including molecular biology research, medical
diagnostics, and even some branches of ecology.

C) Screening of recombinants –Blue white screening
Introduction: Blue-white screening is a rapid and efficient technique for the identification of recombinant
bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose
into glucose and galactose.
Background: The presence of lactose in the surrounding environment triggers the lacZ operon in E. coli.
The operon activity results in the production of β-galactosidase enzyme that metabolizes the lactose. Most
plasmid vectors carry a short segment of lacZ gene that contains coding information for the first 146 amino
acids of β-galactosisdase. The host E. coli strains used are competent cells containing lacZΔM15 deletion
mutation. When the plasmid vector is taken up by such cells, due to α-complementation process, a
functional β-galatosidase enzyme is produced.
The plasmid vectors used in cloning are manipulated in such a way that this α-complementation process
serves as a marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in
the plasmid vector. This sequence can be nicked by restriction enzymes to insert the foreign DNA. When a
plasmid vector containing foreign DNA is taken up by the host E. coli, the α-complementation does not
occur, therefore, a functional β-galactosidase enzyme is not produced. If the foreign DNA is not inserted
into the vector or if it is inserted at a location other than MCS, the lacZ gene in the plasmid vector
complements the lacZ deletion mutation in the host E. coli producing a functional enzyme.
Principle: For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal
is added to the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-
indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-
dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the
recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured.
Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a
non-metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that
IPTG is not a substrate for β-galactosidase but only an inducer. For visual screening purposes, chromogenic
substrate like X-gal is required.

D) Replica plating
This method is used for detection of biochemical mutants, for the classification of fermentation reactions
and for the determination of the spectra of antibiotic sensitivity.
Technique for testing the genetic characteristics of bacterial colonies. A dilute suspension of bacteria is
first spread, in a petri dish, on agar containing a medium expected to support the growth of all bacteria,
the master plate. Each bacterial cell in the suspension is expected to give rise to a colony.
A sterile velvet pad, the same size as the petri dish, is then pressed onto it, picking up a sample of each
colony. The bacteria can then be stamped onto new sterile petri dishes, plates, in
the identical arrangement. The media in the new plates can be made up
to lack specific nutritional requirements or to contain antibiotics. Thus colonies can be identified that
cannot grow without specific nutrients or that are
antibiotic resistant and cells with mutations in particular genes can be isolated.
Principle involved is using the threads of velvet or chamios leather which act as inoculating needles, the
mutants can be replicated and isolated.

E) Northern blotting
 Developed by James Alwine, David Kemp and George Stark in 1977.
 Similarity of name with Southern Blotting but it analyzes RNA
 To study gene expression by detection of RNA during differentiation, diseased conditions
 Electrophoresis – Seperates RNA on the basis of their Molecular weight and type in agarose gel which
have EtBr, an intercalating agent in it.
 Capillary action – RNA bands move towards blotting paper by capillary movement and entrap in sheet
and buffer moves ahead.
 Extraction of RNA From homogenised tissue sample RNA with poly A tail
 Electrophoresis of RNA In agarose gel RNA is not fragmented Transferred to nylon paper or DBM
paper because probes are unable to bind with RNA in gel
 Done by capillary action
 Buffer contains formamide to reduce annealing temp.
 Stabilization by Heat or UV rays Formation of covalent linkages Hybridization With radiolabelled or
fluorescently labelled probe Detection by X-rays

F) Western blotting
Western blotting is a widely used technique for the detection and analysis of proteins based on their ability
to bind to specific antibodies.
It was ﬁrst described by Towbin, et.al in 1979.
Western blotting is an accomplished rapidly, using simple equipment and inexpensive reagents, it is
commonly used laboratory technique.
Western blotting is the transfer of proteins from the SDS- PAGE gel to a solid supporting membrane.
Electrophoresis is used to separate complex mixtures of proteins denaturing discontinuous one
dimensional gel electrophoresis separates proteins only based on molecular size as they move through a
SDS- polyacrylamide gel(SDS PAGE) toward the anode with the smaller protein migrating faster and bigger
proteins running slower.
A protein sample is subjected to polyacrylamide gel electrophoresis. After this the gel is placed over a
sheet of nitrocellulose and the protein in the gel is electrophoretically transferred to the nitrocellulose.
The nitrocellulose is then soaked in blocking buffer (3% skimmed milk solution) to "block" the non-specific
binding of proteins. The nitrocellulose is then incubated with the specific antibody for the protein of
interest. The nitrocellulose is then incubated with a second antibody, which is specific for the first
antibody. The second antibody will typically have a covalently attached enzyme which, when provided
with a chromogenic substrate, will cause a color reaction
Western Blot a Confirmatory test in HIV Infection The virus is enveloped with different proteins. The
detection of these proteins are useful in the detection of the presence of the virus. Western blotting helps
in the detection of these proteins.

G) Southern blotting
Blotting techniques are used to transfer DNA or RNA fragments or proteins from electrophoresis gel to a
nitrocellulose sheet or nylon membrane as blotting paper is used to blot ink. Southern blotting is the
transfer of DNA fragments from an electrophoresis gel to a membranous support which results in
immobilization of DNA fragments. These immobilized single stranded DNA fragments can then be
subjected to hybridization with a labeled probe.
Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh
University in the 1975.
It allows investigators to locate a particular sequence of DNA within a complex mixture. DNA (genomic or
other source) is digested with a restriction enzyme and separated by gel electrophoresis and transferred
from an agarose gel onto a Nitrocellulose sheet or Nylon membrane which is then incubated with a single
stranded DNA probe with known sequence. This probe is supposed to form base pairs with its
complementary DNA sequence and to form a double-stranded DNA molecule. The probe is labeled before
hybridization either radioactively or is treated enzymatically by alkaline phosphatase or horseradish
peroxidase. Finally, the location of hybridization with the probe is detected either by directly exposing the
membrane to X-ray film or by chemiluminescent methods.
STEPS FOR PERFORMING SOUTHERN BLOTTING
• DNA ISOLATION AND PURIFICATION • DIGESTION OF DNA BY RESTRICTION ENDONUCLEASES •
SEPARATE DNA FRAGMENTS BY GEL ELECTROPHOREIS • DS STRANDED DNA FRAGMENTS ON GEL ARE
DENATURED BY ALKALINE TREATMENT TO GIVE SINGLE STRANDED DNA FRAGMENTS  BLOTTING
(TRANSFER OF DNA FRAGMENTS FROM GEL TO NITROCELLULOSE SHEET /NYLON MEMBRANE) 
HYBRIDIZATION OF IMMOBILIZED DNA FRAGMENT WITH SINGLE STRANDED RADIOACTIVELY LABELLED
DNA PROBES  DETECTION OF HYBRIDIZATION BY AUTORADIOGRAPHY OR CHEMILUMNISCENT
METHODS
All of these mutations can be detected by comparing the restriction- fragment-length polymorphisms with
normal fragments of DNA. It is also used to determine the molecular weight of a restriction fragment and

to measure relative amounts in different samples. Under optimal conditions, Southern blotting detects ~
0.1 pg of the DNA of interest.
Detection of the sickle-cell globin gene by Southern blotting: The base change (A T) that causes sickle cell
anaemia a → MstII target site that is present in the normal -globin gene. This difference can be detected by
Southern blotting.
RFLP resulting from -globin gene mutation: β in the normal cell, the sequence corresponding to 5th to 7th
amino acids of the -globin peptide is CCTGAGGAG, which can be recognized by theβ restriction enzyme
MstII. In the sickle cell, one base is mutated from A to T, making the site unrecognizable by MstII. Thus,
MstII will generate 0.2 kb and 1.2 kb fragments in the normal cell, but generate 1.4 kb fragment in the sickle
cell. These different fragments can be detected by the southern blotting.
H) DNA finger printing
DNA-fingerprinting (also called DNA typing or DNA profiling). It is a technique of determining nucleotide
sequences of certain areas of DNA which are unique to each individual. Each person has a unique DNA
fingerprint
Alec Jeffreys (1984) invented the DNA fingerprinting technique at Leicester University, United Kingdom.
Dr. V.K. Kashyap and Dr. Lalji Singh started the DNA fingerprinting technology in India at CCMB (Centre for
Cell and Molecular Biology) Hyderabad.
3,000,000 base pairs of nucleotide that is 0.1% of the genomes are unique in all human being. This
uniqueness in the base sequence doesn't only occur in genes but also in repetitive DNA also known as
satellite DNA. Due to density gradient configuration in the satellite DNA, various small peaks are formed on
the DNA which gives rise to polymorphism. One of the main satellite DNA having high degree of
polymorphism is variable number tandem repeats (VNTR). Since a child receive 50% of the DNA from its
father and the other 50% from his mother, so the number VNTRs at a particular area of the DNA of the child

will be different may be due to insertion, deletion or mutation in the base pairs. As a result, every individual
has a distinct composition of VNTRs and this is the main principle of DNA fingerprinting.
STRs (Short Tandem Repeats) and SSRs (Simple Sequence Repeats)are 2-6 base pair long repeating
sequences. These are unique for every individual and are shorter than VNTRs. Hence they produce
accurate DNA fingerprints.
Applications of DNA Fingerprinting:
DNA fingerprinting has got a lots of applications.
1. This procedure is mostly used in forensic to identify the criminals.
2. It is also used for the paternity test.
3. It is used in the study of breeding patterns of animals facing the danger of extinction.
4. It is also used in determining lineages of humans and other animals to ascertain the process of evolution
by checking the "genetic markers" which are passed from the ancestors.
5. It is used to diagnose the genetic disorders and hereditary disorders like hemophilia, sickle cell anaemia,
cystic fibrosis etc.
6. It is also used to determine about the antibiotics to which bacteria’s are resistant.
I) DNA sequencing- Maxam and Gilbert

J) DNA sequencing- Sanger’s method of sequencing.
Sanger’s method of gene sequencing is also known as dideoxy chain termination method. It generates
nested set of labelled fragments from a template strand of DNA to be sequenced by replicating that
template strand and interrupting the replication process at one of the four bases.
Principle
 A DNA primer is attached by hybridization to the template strand and deoxynucleosides
triphosphates (dNTPPs) are sequentially added to the primer strand by DNA polymerase.
 The primer is designed for the known sequences at 3’ end of the template strand.
 M13 sequences is generally attached to 3’ end and the primer of this M13 is made.
 The reaction mixture also contains dideoxynucleoside triphosphate (ddNTPs) along with usual
dNTPs.
 If during replication ddNTPs is incorporated instead of usual dNTPs in the growing DNA strand then
the replication stops at that nucleotide.
 The ddNTPs are analogue of dNTPs
 ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot make phosphodiester bond
with nest nucleotide, thus terminates the nucleotide chain
 Respective ddNTPs of dNTPs terminates chain at their respective site. For example ddATP
terminates at A site. Similarly ddCTP, ddGTP and ddTTP terminates at C, G and T site respectively.

Procedure
1. Template preparation:
M13-forward-sequence
 Copies of template strand to be sequenced must be prepared with short known sequences at 3’ end
of the template strand.
 A DNA primer is essential to initiate replication of template, so primer preparation of known
sequences at 3’end is always required.
 For this purpose a single stranded cloning vector M13 is flanked with template strand at 3’end which
serves as binding site for primer.
2. Generation of nested set of labelled fragments:
 Copies of each template is divided into four batches and each batch is used for different replication
reaction.
 Copies of standard primer and DNA polymerase I are used in all four batches.
 To synthesize fragments that terminates at A, ddATP is added to the reaction mixture on batch I
along with dATP, dTTP,dCTP and dGTP, standard primer and DNA polymerase I.
Similarly, to generate, all fragments that terminates at C, G and T, the respective ddNTPs ie ddCTP,ddGTP
and ddTTP are added respectively to different reaction mixture on different batch along with usual dNTPs.
3. Electrophoresis and gel reading:
 The reaction mixture from four batches are loaded into four different well on polyacrylamide gel and
electrophoresed.
 The autoradiogram of the gel is read to determine the order of bases of complementary strand to
that of template strand.
 The band of shortest fragments are at the bottom of autoradiogram so that the sequences of
complementary strand is read from bottom to top.

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