B.

Tech Bio-Technology

IInd Year IVth Semester

Genetic & Molecular Biology
Lab Manual
 Experiment 1: To Isolate Plasmid DNA from Escherichia coli (E. coli) Using the
Alkaline Lysis Method

AIM

The aim of this experiment is to isolate plasmid DNA from Escherichia coli (E. coli) using the
alkaline lysis method. This procedure enables the selective extraction of plasmid DNA from
bacterial cells by exploiting differences in size and topology between chromosomal and plasmid
DNA. The isolated DNA should be of sufficient quality and quantity for downstream molecular
biology applications, such as restriction digestion, PCR amplification, cloning, and sequencing.

MATERIALS REQUIRED

A. Bacterial Culture

 Overnight-grown E. coli culture in LB broth containing the plasmid of interest.

B. Reagents

 Solution I (Resuspension Buffer):

o 50 mM Glucose
o 25 mM Tris-HCl (pH 8.0)
o 10 mM EDTA
 Solution II (Lysis Buffer):
o 0.2 N NaOH (sodium hydroxide)
o 1% SDS (sodium dodecyl sulfate)
 Solution III (Neutralization Buffer):
o 3 M Potassium acetate, pH adjusted to 4.8 with acetic acid
 RNase A (10 mg/ml)
 Isopropanol (room temperature or chilled)
 70% Ethanol (cold)
 TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
 C. Equipment and Consumables

 Microcentrifuge tubes (1.5 ml)

 Microcentrifuge capable of 12,000 rpm
 Vortex mixer
 Pipettes and sterile tips
 Water bath or incubator set at 37°C
 Ice bath
 Micropestle (if needed for cell disruption)
 UV spectrophotometer or Nanodrop (optional, for quantification)
 Agarose gel electrophoresis setup (optional, for visualization)

PRINCIPLE

The alkaline lysis method selectively isolates plasmid DNA from bacterial chromosomal DNA
by:

1. Lysing the cells in a highly alkaline environment which denatures both chromosomal and
plasmid DNA.
2. Neutralizing the solution to allow plasmid DNA to reanneal correctly (due to its small, circular,
supercoiled structure) while large chromosomal DNA remains denatured and precipitates out.
3. Centrifugation removes the precipitated chromosomal DNA and cell debris, leaving plasmid
DNA in the supernatant.
4. Precipitation of plasmid DNA with alcohol (isopropanol), followed by ethanol washing and
resuspension.

PROCEDURE

Step 1: Preparation of Bacterial Pellet

1. Inoculate a single colony of E. coli containing the plasmid into 5 ml of LB broth and incubate
overnight at 37°C with vigorous shaking (~180 rpm).
2. Transfer 1.5 ml of the culture into a 1.5 ml microcentrifuge tube.
3. Centrifuge at 12,000 rpm for 1 minute at room temperature to pellet the cells.
4. Carefully discard the supernatant without disturbing the pellet.

Step 2: Cell Resuspension

5. Resuspend the pellet completely in 100 µl of Solution I by pipetting up and down or vortexing
gently. This buffer helps to stabilize the DNA and protect it from degradation by chelating
divalent cations (via EDTA).
6. Add 2 µl of RNase A to degrade RNA contaminants and mix thoroughly.

Step 3: Cell Lysis

7. Add 200 µl of freshly prepared Solution II to the tube.

8. Gently invert the tube 4–6 times to mix. DO NOT VORTEX, as this can shear chromosomal
DNA, causing contamination of your plasmid prep.
9. Incubate the tube on ice for 5 minutes. The solution will become clear and viscous as the cells
lyse and DNA denatures.

Step 4: Neutralization

10. Add 150 µl of Solution III (cold potassium acetate buffer, pH 4.8) to neutralize the alkaline
environment.
11. Immediately and gently invert the tube 4–6 times. A white flocculent precipitate will form, which
includes denatured chromosomal DNA, proteins, and cell debris.
12. Incubate on ice for 5–10 minutes to enhance precipitation.

Step 5: Separation of Plasmid DNA

13. Centrifuge the tube at 12,000 rpm for 10 minutes at 4°C.

14. Carefully transfer the clear supernatant (containing plasmid DNA) to a fresh microcentrifuge
tube, avoiding the white pellet.
 Step 6: DNA Precipitation

15. Add 0.6 volumes of isopropanol to the recovered supernatant (e.g., add 210 µl isopropanol to
350 µl supernatant).
16. Mix gently by inverting the tube.
17. Incubate at room temperature for 10 minutes or on ice for 30 minutes to allow DNA
precipitation.

Step 7: DNA Recovery

18. Centrifuge at 12,000 rpm for 10 minutes. A small, white DNA pellet should appear at the bottom
of the tube.
19. Carefully discard the supernatant.
20. Add 500 µl of cold 70% ethanol to wash the DNA pellet. Invert gently.
21. Centrifuge at 12,000 rpm for 5 minutes, then discard the ethanol carefully.
22. Air-dry the pellet by leaving the tube open at room temperature for 10–15 minutes. Do not
overdry.
23. Resuspend the pellet in 30–50 µl of TE buffer by pipetting or gentle vortexing.

OBSERVATION

 After precipitation and centrifugation, a small white pellet of plasmid DNA is observed.
 Upon running 5 µl of the resuspended DNA on a 0.8–1% agarose gel stained with ethidium
bromide or SYBR Safe:
o A distinct, sharp band is visible under UV light.
o The band often shows supercoiled, open circular, and possibly nicked circular forms of
plasmid DNA.

RESULT

Plasmid DNA was successfully isolated from E. coli using the alkaline lysis method. The
resulting DNA was:
 Visibly present as bands under UV light after gel electrophoresis.
 Free of significant RNA or protein contamination.
 Suitable for downstream applications such as cloning, PCR amplification, and restriction
digestion.

PRECAUTIONS

 Ensure aseptic techniques throughout the experiment to avoid contamination.

 Do not vortex after adding Solution II to avoid breaking chromosomal DNA into fragments that
could contaminate the plasmid prep.
 Use freshly prepared reagents, particularly RNase A and potassium acetate buffer.
 Be cautious while removing the supernatant after centrifugation — avoid disturbing the pellet.
 Ethanol and isopropanol are flammable; handle with care and keep away from flame or heat
sources.
 Always wear gloves and safety goggles when handling ethidium bromide (or use a safer dye).
 Do not allow the DNA pellet to overdry, as this may make it difficult to resuspend.
 Experiment 2: To Isolate Genomic DNA from Plant Tissue (e.g., Spinach or Onion)
using the CTAB Method

AIM

To isolate high-quality, high molecular weight genomic DNA from plant tissues such as
spinach or onion using the CTAB (Cetyl Trimethyl Ammonium Bromide) method, which
effectively removes polysaccharides and polyphenols — common contaminants in plant DNA
extractions — and provides DNA suitable for molecular biology applications.

FULL FORM OF CTAB

CTAB – Cetyl Trimethyl Ammonium Bromide

A cationic detergent used to lyse plant cells by disrupting the cell membrane and to selectively
precipitate polysaccharides during DNA extraction, thus facilitating the isolation of pure DNA.

MATERIALS REQUIRED

A. Plant Material

 ~0.5 g of fresh, young spinach or onion leaves (tender tissue is preferred as it yields more intact
DNA and fewer contaminants).

B. Reagents

 CTAB Extraction Buffer (pre-warmed to 65°C):

o 2% CTAB
o 100 mM Tris-HCl (pH 8.0)
o 20 mM EDTA
o 1.4 M NaCl
o 0.2% β-mercaptoethanol (add freshly before use)
 Chloroform:Isoamyl alcohol (24:1) – for phase separation
 Isopropanol (cold) – for DNA precipitation
 70% Ethanol (cold) – for washing DNA pellet
 TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) – for DNA resuspension
 Liquid nitrogen – for efficient grinding and cell disruption (optional but recommended)

C. Equipment

 Mortar and pestle (preferably pre-chilled)

 Water bath or incubator (set to 65°C)
 Microcentrifuge and 1.5 ml centrifuge tubes
 Ice bucket
 Micropipettes and sterile tips
 Vortex mixer
 Spectrophotometer or Nanodrop (optional for DNA quantification)
 Agarose gel electrophoresis apparatus (optional for visualization)

PRINCIPLE

The CTAB method is specifically designed for plant tissues which are rich in secondary
metabolites, polysaccharides, and polyphenolic compounds that interfere with DNA isolation.
CTAB forms complexes with polysaccharides and helps in their removal during extraction. A
high salt concentration (NaCl) ensures proteins and polysaccharides remain in solution or form
complexes that can be separated during chloroform extraction. The DNA is selectively
precipitated using isopropanol and purified by ethanol washing.

PROCEDURE

Step 1: Tissue Homogenization

1. Weigh approximately 0.5 g of fresh plant leaves (spinach/onion).

2. Place the leaves in a pre-chilled mortar and add liquid nitrogen.
3. Grind thoroughly with a pestle until a fine powder is obtained (this step breaks the tough cell
wall and prevents enzymatic degradation by DNases).

Step 2: Cell Lysis

4. Quickly transfer the powdered sample into a 1.5 ml centrifuge tube containing 700 µl of pre-
warmed CTAB extraction buffer (65°C).
5. Mix gently but thoroughly to ensure complete contact between buffer and sample.
6. Incubate at 65°C for 30 minutes with intermittent mixing. This allows for cell lysis,
denaturation of proteins, and binding of DNA to CTAB, while secondary metabolites begin to
separate.

Step 3: Phase Separation

7. Cool the tubes to room temperature.

8. Add 700 µl of chloroform:isoamyl alcohol (24:1) to the lysed mixture.
9. Gently invert the tubes for 5–10 minutes to allow mixing and denaturation of proteins.
10. Centrifuge at 12,000 rpm for 10 minutes at room temperature to form two distinct phases:
o The upper aqueous layer contains the DNA.
o The interphase and lower organic layer contain proteins, lipids, and cell debris.

Step 4: DNA Precipitation

11. Carefully transfer the clear upper aqueous layer into a fresh tube using a pipette. Avoid
disturbing the interface.
12. Add 0.6 to 0.7 volume of cold isopropanol (e.g., 420–490 µl isopropanol for 700 µl supernatant)
to precipitate DNA.
13. Mix by gently inverting several times.
14. Incubate the mixture at –20°C for 20–30 minutes or at room temperature for 10 minutes.

Step 5: DNA Recovery

15. Centrifuge the mixture at 12,000 rpm for 10 minutes to pellet the DNA.
16. Carefully discard the supernatant.
17. Wash the DNA pellet with 500 µl of cold 70% ethanol to remove residual salts and impurities.
18. Centrifuge again at 12,000 rpm for 5 minutes.
19. Discard the ethanol and air-dry the DNA pellet for 10–15 minutes at room temperature (do not
over-dry).

Step 6: DNA Resuspension

20. Resuspend the DNA pellet in 50–100 µl of TE buffer.

21. Incubate at 37°C for 30 minutes to ensure complete dissolution of DNA.

OBSERVATION

 After precipitation, a whitish, stringy or flaky DNA pellet should be visible.

 When the DNA is run on a 0.8–1% agarose gel, you should observe:
o A thick, high-molecular-weight DNA band near the well.
o Minimal smearing (indicates less fragmentation).
 The DNA sample should have a 260/280 nm absorbance ratio of ~1.8, indicating purity (if
measured using a spectrophotometer).

RESULT

Genomic DNA was successfully extracted from spinach/onion leaves using the CTAB
extraction method. The DNA appeared as a clear pellet and was visualized as intact bands on
agarose gel electrophoresis. The extracted DNA is of good yield, integrity, and purity, making it
suitable for downstream molecular applications such as PCR, restriction digestion, RAPD, AFLP,
and sequencing.

PRECAUTIONS

 Use young and tender leaves as older tissues contain more polysaccharides and polyphenols.
 Add β-mercaptoethanol fresh to CTAB buffer as it prevents oxidation of phenolic compounds.
 Avoid disturbing the interface during phase separation to prevent protein contamination.
 Always work on ice or chilled conditions to preserve DNA integrity.
 Use RNase if RNA contamination is not desired.
 Handle chloroform and β-mercaptoethanol in a fume hood and wear gloves as both are
hazardous.
 Ensure all glassware and plasticware is nuclease-free.
 Avoid overdrying the DNA pellet, as it makes resuspension difficult and may shear DNA.
 If DNA is to be stored long-term, keep at –20°C in TE buffer.
 Experiment 3: Determination of Tm (Melting Temperature) of DNA

AIM

To determine the melting temperature (Tm) of DNA using UV absorbance spectroscopy by

monitoring the increase in absorbance at 260 nm with rising temperature, which reflects the
denaturation of double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA). Tm
provides information about the thermal stability of DNA and its sequence composition.

PRINCIPLE

DNA absorbs ultraviolet (UV) light most efficiently at 260 nm due to the presence of nitrogenous
bases (adenine, guanine, cytosine, thymine). In double-stranded DNA, the bases are stacked and
hydrogen-bonded, reducing absorbance (hypochromic effect). As temperature increases, hydrogen
bonds between base pairs break, and DNA strands separate (denature), exposing bases to the
solvent, leading to increased absorbance (hyperchromic effect).

This change in absorbance can be plotted against temperature. The melting temperature (Tm) is
defined as the temperature at which 50% of the DNA becomes single-stranded, i.e., the
midpoint of the absorbance vs. temperature sigmoidal curve.

MATERIALS REQUIRED

Reagents

 DNA sample (e.g., calf thymus DNA, λ-DNA, or purified genomic/plasmid DNA)
 Phosphate buffer or SSC buffer (pH 7.0) to maintain ionic strength and pH stability
 Distilled or deionized water

Equipment

 UV-Visible Spectrophotometer equipped with a temperature control unit

 Quartz cuvette (1 ml or 500 µl volume; plastic cuvettes are not UV-compatible)
 Water bath or Peltier-controlled heating system (to adjust temperature precisely)
 Thermometer or digital probe (to confirm internal cuvette temperature)
 Micropipettes and sterile tips
 Ice bath (for pre- and post-heating sample control)

PROCEDURE

Step 1: DNA Sample Preparation

1. Dilute the DNA sample to a final concentration of approximately 50 µg/ml using phosphate
buffer (pH 7.0).
2. Mix gently to avoid shearing the DNA.
3. Transfer 1 ml of the sample into a clean quartz cuvette using a micropipette.
4. Seal the cuvette to avoid evaporation during heating.

Step 2: Baseline Absorbance Measurement

5. Set the spectrophotometer to 260 nm wavelength.

6. Measure and record the initial absorbance at 25°C, ensuring no air bubbles are present.
7. This reading will serve as the baseline (A₀).

Step 3: Temperature Ramp and Absorbance Monitoring

8. Gradually increase the temperature in 5°C increments starting from 25°C up to ~95°C.
9. At each temperature step:
o Wait for 2–3 minutes to allow the sample to reach thermal equilibrium.
o Record the absorbance at 260 nm.
10. Continue this process until the absorbance plateaus, indicating complete DNA denaturation.
 Step 4: Optional Cooling

11. Optionally, cool the sample back to 25°C and observe whether a reduction in absorbance
occurs, indicating renaturation (re-annealing) of DNA.

Step 5: Data Analysis and Plotting

12. Plot a graph of Absorbance at 260 nm (Y-axis) vs Temperature (°C) (X-axis).

13. The plot will display a sigmoidal (S-shaped) curve.
14. Identify the midpoint of the transition curve — this is the melting temperature (Tm), where
half of the DNA molecules are denatured.

OBSERVATION

 A gradual increase in absorbance at 260 nm is observed with increasing temperature.

 The curve shows a sharp rise in absorbance near the melting region, followed by a plateau.
 The Tm is usually found in the range of 70–90°C, depending on the DNA's base composition
(higher GC content = higher Tm).

RESULT

The melting temperature (Tm) of the DNA sample was successfully determined using UV
absorbance spectroscopy. The Tm indicates the temperature at which half of the DNA is
denatured. For example, a typical calf thymus DNA sample may exhibit a Tm around 72°C,
which suggests moderate GC content and standard ionic strength.

FACTORS AFFECTING Tm

 GC content: Guanine-cytosine pairs have three hydrogen bonds (vs two in A–T), increasing Tm.
 Ionic strength: Higher salt concentrations stabilize the double helix, raising Tm.
 DNA length: Longer fragments tend to have higher Tm.
 pH of buffer: Extreme pH values can denature DNA or affect base ionization.
 Mismatches in base pairing: Lower Tm due to reduced duplex stability.

PRECAUTIONS

 Always use quartz cuvettes for UV readings to avoid inaccurate results.

 Ensure the DNA solution is free of contaminants (RNA, proteins, phenol) as they may absorb at
260 nm and skew results.
 Avoid air bubbles in the cuvette — they can cause light scattering and absorbance artifacts.
 Mix DNA thoroughly but gently to prevent shearing.
 Handle cuvettes carefully — keep them clean and dry, free of scratches or fingerprints.
 Calibrate the spectrophotometer and temperature control unit before use.
 Use freshly prepared DNA to avoid degraded samples, which could affect Tm.

Experiment 4: Isolation of Bacterial Genomic DNA

AIM

To isolate high molecular weight genomic DNA from bacterial cells (e.g., Escherichia coli)
using enzymatic digestion, detergent-based cell lysis, organic extraction, and alcohol
precipitation. This procedure is intended to yield DNA of sufficient purity and quantity for
downstream molecular biology applications such as PCR, restriction digestion, and sequencing.

PRINCIPLE

Genomic DNA isolation from bacteria involves several key steps:

1. Lysis of the bacterial cell wall and membrane using a combination of lysozyme, SDS, and
Proteinase K.
2. Removal of RNA using RNase A.
3. Separation of DNA from proteins and lipids through phenol:chloroform extraction.
4. DNA precipitation using cold isopropanol, followed by washing with 70% ethanol.
5. Resuspension of the DNA pellet in TE buffer or sterile water for storage and use.

The result is intact, purified genomic DNA free from RNA, proteins, and other cellular
contaminants.

MATERIALS REQUIRED

A. Bacterial Culture

 E. coli or any bacterial strain grown overnight in Luria-Bertani (LB) broth at 37°C with shaking
(180–220 rpm).

B. Reagents

 TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) – stabilizes and protects DNA

 Lysis Buffer – typically TE buffer with 1% SDS
 Lysozyme (optional for Gram-negative, essential for Gram-positive; 10 mg/ml)
 Proteinase K (20 mg/ml) – digests proteins
 RNase A (10 mg/ml) – degrades RNA contaminants
 Phenol:Chloroform:Isoamyl alcohol (25:24:1) – removes proteins and lipids
 Chloroform:Isoamyl alcohol (24:1) – removes residual phenol
 Cold Isopropanol – precipitates DNA
 70% Cold Ethanol – washes away salts and impurities
 Sterile distilled water or TE buffer – for resuspension of DNA

C. Equipment

 Microcentrifuge and 1.5 ml microcentrifuge tubes

 Vortex mixer
 Water bath or incubator (set at 37°C and 55°C)
 Ice bucket
 Micropipettes and sterile tips
 Optional: Nanodrop or UV spectrophotometer
 Optional: Agarose gel electrophoresis unit

PROCEDURE

🔹 Step 1: Culture and Cell Harvesting

1. Inoculate 3–5 ml LB broth with a single colony of E. coli and incubate overnight at 37°C with
shaking.
2. Transfer 1.5 ml of the culture into a microcentrifuge tube.
3. Centrifuge at 12,000 rpm for 1 minute to pellet the cells.
4. Discard the supernatant and wash the pellet with 500 µl TE buffer. Centrifuge again and discard
the wash.

Step 2: Cell Wall and Membrane Lysis

5. Resuspend the pellet in 200 µl TE buffer.

6. Add 20 µl of lysozyme (10 mg/ml) and incubate at 37°C for 15 minutes to digest the bacterial
cell wall.
7. Add 20 µl of SDS (10%) to solubilize the cell membrane.
8. Add 10 µl of Proteinase K (20 mg/ml) to digest cellular proteins.
9. Incubate the mixture at 55°C for 1 hour or until the lysate becomes clear, indicating cell lysis.

Step 3: RNA Removal

10. Add 10 µl of RNase A (10 mg/ml) and incubate at 37°C for 30 minutes to degrade RNA.

Step 4: Organic Extraction

11. Add 500 µl of phenol:chloroform:isoamyl alcohol (25:24:1) to the lysate.

12. Mix gently by inversion for 5–10 seconds. Do not vortex to avoid shearing genomic DNA.
13. Centrifuge at 12,000 rpm for 10 minutes to separate the phases.
14. Carefully transfer the upper aqueous layer (contains DNA) into a new microcentrifuge tube.
15. Add an equal volume of chloroform:isoamyl alcohol (24:1) to the aqueous phase.
16. Mix gently and centrifuge again at 12,000 rpm for 10 minutes.
17. Transfer the upper phase to a fresh tube.

Step 5: DNA Precipitation

18. Add 0.6 volume of cold isopropanol to the DNA-containing aqueous layer (e.g., 300 µl
isopropanol to 500 µl aqueous layer).
19. Gently invert to mix and incubate at –20°C for 30 minutes to allow DNA precipitation.

Step 6: Washing and Resuspension

20. Centrifuge at 12,000 rpm for 10 minutes. A white DNA pellet should form.
21. Carefully discard the supernatant and add 500 µl of 70% cold ethanol to wash the pellet.
22. Centrifuge again for 5 minutes at 12,000 rpm.
23. Remove ethanol, air-dry the pellet for 10–15 minutes (do not overdry).
24. Resuspend the DNA in 50–100 µl of TE buffer or sterile distilled water.
25. Store the DNA at –20°C for long-term use.

OBSERVATION

 After precipitation and centrifugation, a white or off-white DNA pellet should be visible.
 When visualized on 0.8–1% agarose gel:
o A single, high-molecular-weight DNA band is expected near the top of the gel.
 A Nanodrop reading or spectrophotometer may show:
o A260/A280 ratio of ~1.8, indicating pure DNA with minimal protein contamination.

RESULT

Genomic DNA was successfully isolated from Escherichia coli using enzymatic digestion and
organic solvent extraction. The DNA was:
 High in molecular weight
 Free from significant RNA and protein contamination
 Suitable for downstream molecular biology applications such as PCR, restriction digestion, or
sequencing

PRECAUTIONS

 Use ice or chilled conditions during extraction to minimize DNase activity.

 Do not vortex after SDS addition, as this may shear genomic DNA.
 Use clean, nuclease-free equipment to avoid DNA degradation.
 Avoid phenol contamination in the final DNA by proper phase separation.
 Use RNase to eliminate RNA that may interfere with downstream applications.
 Handle phenol and chloroform with care — they are toxic and volatile; always work under a
fume hood and wear gloves.
 Do not overdry DNA pellets as they become difficult to dissolve.
 If working with Gram-positive bacteria, increase lysozyme concentration and incubation time
due to the thick peptidoglycan layer.
 Experiment 5: To Separate DNA Fragments Based on Their Size Using
Polyacrylamide Gel Electrophoresis (PAGE)

AIM

To separate DNA fragments according to their size using polyacrylamide gel electrophoresis
(PAGE), a high-resolution technique best suited for analyzing short DNA fragments (10–1000
base pairs). PAGE offers greater resolution than agarose gel, making it ideal for applications like
oligonucleotide analysis, DNA sequencing, and mutation detection.

PRINCIPLE

DNA molecules are negatively charged due to their phosphate backbone and migrate towards the
positive electrode when placed in an electric field. In polyacrylamide gels, the matrix acts as a
molecular sieve, separating DNA fragments based on size. Smaller fragments migrate faster and
more easily through the pores of the gel, while larger fragments move slower.

Polyacrylamide gels are preferred over agarose for small DNA fragment separation due to:

 Smaller pore size allowing fine resolution

 Tighter bands and sharper visualization
 Useful in detecting single base differences
 MATERIALS REQUIRED

A. Reagents

 DNA Sample: Purified or digested DNA, synthetic oligonucleotides

 29:1 Acrylamide:Bisacrylamide (30%): The polymer forming the gel matrix
 10X TBE Buffer: Provides conductivity and maintains pH (Tris, Boric Acid, EDTA)
 APS (Ammonium Persulfate, 10%): Initiates gel polymerization
 TEMED: Catalyzes free-radical formation to complete polymerization
 6X DNA Loading Dye: Contains tracking dye and glycerol for sample density
 Ethidium Bromide or SYBR Safe: DNA intercalating dye for visualization
 DNA Ladder/Marker: Standard for fragment size estimation
 Distilled Water: For dilution and cleaning

B. Equipment

 PAGE Gel Casting Assembly: Includes glass plates, spacers, and combs
 Vertical Electrophoresis Tank and Power Supply
 Micropipettes and Sterile Tips
 UV Transilluminator or Gel Documentation System
 Water Bath or Heat Block (for DNA denaturation, if required)
 Protective Gear: Gloves, lab coat, and safety goggles

PROCEDURE

Step 1: Gel Preparation

1. Decide the percentage of gel:

o 6–8% for ~500–1000 bp
o 10–12% for 100–500 bp
o 15% for <100 bp
2. To prepare 10 ml of 12% polyacrylamide gel:
o 4 ml 30% acrylamide:bisacrylamide
o 1 ml 10X TBE buffer
o 4.9 ml distilled water
o 100 µl 10% APS (fresh)
o 10 µl TEMED
3. Quickly pour gel between glass plates, insert comb, and allow to polymerize for 30–45 minutes.

Step 2: Sample Preparation

4. Mix 5 µl DNA sample with 1 µl of 6X loading dye.

5. If denaturation is needed (e.g., for single-stranded DNA or tightly bound structures):
o Heat the sample at 95°C for 3–5 minutes, then immediately cool on ice.
6. Briefly spin to collect the contents.

Step 3: Loading and Running the Gel

7. After polymerization, gently remove the comb.

8. Place the gel into the vertical electrophoresis chamber and fill with 1X TBE buffer.
9. Load:
o DNA samples
o DNA ladder/marker in one lane for size comparison
10. Run the gel at 80–120 V for 1–2 hours until the tracking dye nears the bottom.

Step 4: Staining (If Not Pre-stained)

11. Immerse the gel in ethidium bromide (0.5 µg/ml) or SYBR Safe for 15–30 minutes.
12. Destain in distilled water to reduce background (if necessary).

Step 5: Visualization

13. Place the gel on a UV transilluminator or inside a gel doc system.

14. Observe and capture the image of the fluorescent DNA bands.
15. Compare the bands with the DNA ladder to estimate fragment sizes.

OBSERVATION

 DNA fragments appear as discrete, sharp bands.

 Smaller fragments migrate faster and move further down the gel.
 Larger fragments migrate slower, staying closer to the wells.
 The band position correlates with molecular size using the DNA ladder.

RESULT

DNA fragments were successfully separated based on their size using PAGE. The gel displayed
clear banding patterns, and the migration distances of the fragments could be compared with the
DNA ladder to estimate their molecular weight.

PRECAUTIONS

 Handle unpolymerized acrylamide with care; it is a neurotoxin.

 Use fresh APS and TEMED for effective and uniform polymerization.
 Ensure gel plates are clean and sealed tightly to prevent leakage.
 Avoid air bubbles during gel casting — they interfere with uniform migration.
 Ensure equal loading volumes across wells for consistent results.
 Always use proper PPE when handling ethidium bromide or work with safer alternatives like
SYBR Safe.
 Run the gel vertically and evenly to avoid curved bands.
 Select the appropriate gel percentage based on expected fragment sizes for best resolution.

Applications of PAGE in DNA Studies

 Accurate sizing of PCR products or restriction fragments

 Detection of mutations or SNPs
 DNA footprinting or binding assays
 DNA sequencing gels
 High-resolution separation for microsatellite or SSR analysis
Experiment 6: To Amplify a Specific DNA Region Using Polymerase Chain Reaction
(PCR) and Visualize the Amplified DNA by Gel Electrophoresis

AIM

To selectively amplify a specific region of DNA using the Polymerase Chain Reaction (PCR),
a molecular technique that enables exponential replication of DNA segments in vitro. The
 experiment also aims to confirm the success and accuracy of amplification by analyzing the PCR
product through agarose gel electrophoresis.

PRINCIPLE

PCR is a revolutionary technique developed by Kary Mullis that allows for rapid, repeated, and
specific duplication of a target DNA sequence using:

 A template DNA
 Two specific primers (forward and reverse)
 Taq DNA polymerase (a heat-stable enzyme)
 dNTPs (building blocks)
 Buffer with Mg²⁺ ions

The PCR involves three major steps, repeated for multiple cycles:

1. Denaturation (94–95°C): Melts double-stranded DNA into single strands.

2. Annealing (50–60°C): Primers bind to their complementary sequences on the template.
3. Extension (72°C): Taq polymerase synthesizes new DNA strands from the primers.

Each cycle doubles the number of DNA molecules, resulting in exponential amplification.

Following PCR, the size and presence of the amplified DNA is verified using agarose gel
electrophoresis, which separates DNA based on fragment size.

MATERIALS REQUIRED

A. Reagents

 Template DNA: Genomic DNA or plasmid DNA containing the target region.
 Forward Primer (10 µM): Short oligonucleotide complementary to the 5' end of the target.
 Reverse Primer (10 µM): Complementary to the 3' end.
 dNTP Mix (10 mM): A mix of all four deoxynucleotide triphosphates.
 10X PCR Buffer with MgCl₂: Provides optimal conditions for Taq polymerase.
 Taq DNA Polymerase (5 U/µl): Thermostable enzyme from Thermus aquaticus.
 Nuclease-free water: To bring the reaction volume to 25 µl.
 DNA loading dye: For loading and tracking migration on gel.
 Agarose powder
 Ethidium bromide or SYBR Safe (for staining DNA)
 DNA Ladder/Marker (100 bp or 1 kb, depending on expected product)

B. Equipment

 Thermal cycler (PCR machine)

 Micropipettes and filter tips
 PCR tubes (0.2 ml)
 Agarose gel electrophoresis unit
 Power supply
 UV transilluminator or Gel documentation system
 Water bath or heat block (optional for hot-start reactions)

PROCEDURE

Step 1: PCR Reaction Setup

1. Thaw all reagents on ice and briefly vortex and spin down.
2. Prepare a PCR master mix for multiple reactions to minimize pipetting errors.
A standard 25 µl reaction contains:
o 2.5 µl of 10X PCR buffer
o 0.5 µl of dNTP mix (10 mM each)
o 0.5 µl of forward primer (10 µM)
o 0.5 µl of reverse primer (10 µM)
o 0.2 µl of Taq DNA polymerase (5 U/µl)
o 1–2 µl of template DNA (~50 ng)
o Sterile water to bring the volume to 25 µl
3. Dispense into PCR tubes and cap tightly.
4. Briefly spin down the tubes and place them in the thermal cycler.

Step 2: PCR Cycling Conditions

Set the thermal cycler with the following program (adjust temperatures for your primer set):

 Initial Denaturation: 94°C for 3 minutes

 30 Cycles of:
o Denaturation: 94°C for 30 seconds
o Annealing: 50–60°C for 30 seconds (exact temperature depends on primer Tm)
o Extension: 72°C for 1 minute (1 minute per kb of target)
 Final Extension: 72°C for 5 minutes
 Hold: 4°C (until analysis)

Step 3: Agarose Gel Electrophoresis

1. Prepare a 1–1.5% agarose gel in 1X TAE or TBE buffer and allow it to solidify in a gel casting
tray.
2. Add ethidium bromide (0.5 µg/ml) or use pre-cast SYBR Safe.
3. Mix 5 µl of PCR product with 1 µl of loading dye.
4. Load the sample into wells along with a DNA ladder.
5. Run the gel at 80–120 V for 30–45 minutes.
6. View bands under a UV transilluminator and document the image.

OBSERVATION

 A sharp DNA band appears at the position correlating with the expected size of the PCR
product.
 No band in the negative control lane indicates the absence of contamination.
 The DNA ladder provides a reference for size estimation.
 RESULT

The desired DNA fragment was successfully amplified using PCR and confirmed by agarose gel
electrophoresis. The DNA band matched the expected size, indicating that:

 Primers were specific

 Reaction components were effective
 No significant contamination occurred

PRECAUTIONS

 Use nuclease-free water and sterile tubes/tips to prevent contamination.

 Always set up a negative control (no DNA) to monitor for contamination.
 Perform pre- and post-PCR procedures in separate areas.
 Design primers with appropriate melting temperatures (Tm) and avoid self-complementarity.
 Avoid repeated freeze-thaw cycles of enzymes and dNTPs.
 Do not open PCR tubes post-amplification near the setup area to avoid aerosol contamination.
 Handle ethidium bromide with gloves and dispose of it as hazardous waste.

Applications of PCR

 Disease diagnosis (e.g., viral and bacterial infections)

 Genetic fingerprinting and forensics
 Gene cloning
 Mutation analysis
 Quantitative PCR (qPCR) for gene expression studies