1.
Dr. Rajendra Prasad Central Agricultural University, BIHAR
PUSA ( Samastipur ) – 848125
COURSE : BAB – 504 (Techniques in molecular & cell biology)
Department of Agricultural Biotechnology &
molecular biology
Assignment
on
PCR – RFLP Analysis
Submitted to Submitted by
Dr. Rajeev Kumar Vishnu Kumar
Asstt. Prof., AB & MB MSc. Agri. Bioteh

2.
Restriction Fragment Length Polymorphism Analysis of
(PCR-RFLP) and Gel Electrophoresis
MAJOR STEPS :
1. DNA isolation and purification
2. PCR amplification and restriction enzyme digestion
3. Separation and detection of the digested products via electrophoresis
DNA Extraction Protocol
Introduction
Plant materials are among the most difficult for high quality
DNA extractions. The key is to properly prepare the tissues for
extraction. In most cases this involves the use of liquid nitrogen flash
freezing followed by grinding the frozen tissue with a mortar and
pestle. Liquid nitrogen is difficult to handle and it is dangerous in an
open laboratory environment such as a classroom. For this reason we
have modified a very simple plant DNA extraction protocol to use
fresh tissue. For isolation of genomic DNA, 7-10 days old seedling
will be used by using CTAB (Khan et al., 2004 ) method adopted from
Doyle & Doyle (1987) with appropriate modifications.

3.
Reagents and Buffers
Extraction Buffer A (EBA) Per 100 mL
2% (w/v) hexadecyltrimethylammonium bromide (CTAB) 2.0 g
100 mM Tris (pH 8.0) (Use 1 M stock) 10 mL
20 mM EDTA (Use 0.5 M stock) 1 mL
1.4 M NaCl 8.2 g
4% (w/v) polyvinylpyrrolidone (PVP) 4.0 g
0.1% (w/v) ascorbic acid 0.1 g
10 mM β-mercaptoethanol (BME)* (Use 14.3 M stock) 70 μL
Extraction Buffer B (EBB) Per 100 mL
100 mM Tris-HCl (pH 8.0) (Use 1 M stock) 10 mL
50 mM EDTA (Use 0.5 M stock) 2.5 mL
100 mM NaCl 0.6 g
10 mM β-mercaptoethanol (BME)* (Use 14.3 M stock) 70 μL
TE Buffer Per 100 mL
10 mM Tris (pH 8.0) (Use 1 M stock) 1.0 mL
1 mM EDTA (Use 0.5 M stock) 50 μL
Other Required Reagents
20% (w/v) sodium dodecyl sulphate (SDS)
5 M potassium acetate (Stored at –20oC)
3 M sodium acetate (pH 5.2)
70% ethanol (stored at -20oC)
Absolute isopropanol (stored at -20oC)

4.
Extraction Protocol
1. Weight out 0.3 g of plant tissue
2. Place tissue on a clean glass slide. Chop the tissue into a paste
using a clean single edge razor blade.
3. Immediately transfer tissue to a 1.5 mL microcentrifuge tube and
further grind tissue with a tube pestle
4. Once the sample is prepared add 300 μL EBA, 900μl EBB, and
100 μl SDS.
5. Vortex and incubate at 65oC for 10 min.
6. Place tube on ice and add 410 μL cold potassium acetate. Mix by
inversion and place tube back on ice for 3 min.
7. Centrifuge at 13,200 rpm for 15 min. (If possible, use a
refrigerated micro-centrifuge set to 4oC)
8. Transfer 1 mL of the supernatant to a new 1.5 mL
microcentrifuge tube, add 540 μL of ice cold absolute
isopropanol, and incubate in ice for 20 min.
9. Centrifuge at 10,200 rpm for 10 min. discard the supernatant.
Wash the pellet once in 500 μL 70% ethanol and let dry
10. Resuspend the dry pellet in 600 μL of TE. Add 60 μL 3M
sodium acetate (pH 5.2) and 360 μL ice cold absolute
isopropanol. Incubate on ice for 20 min.
11. Repeat Steps 9−11 twice.
12. Resuspend the pellet in 50 μL TE and carry out agarose gel
electrophoresis.

5.
PCR –RFLP PROTOCOL
INTRODUCTION :
• Isolation of sufficient DNA for RFLP analysis is time-
consuming and labor intensive. However, PCR can be used to
amplify very small amounts of DNA, usually in 2-3 hours, to the
levels required for RFLP analysis. Therefore, more samples can
be analyzed in a shorter time.
• PCR-RFLP consists of several separate steps including design of
primers, identification of an appropriate restriction enzyme,
amplification, restriction enzyme treatment of amplified
products and electrophoresis to resolve the restriction fragments.
• Below a description of PCR-RFLP is provided. In this
description attention will be focused upon the design of primers,
identification of appropriate restriction enzymes and
electrophoretic techniques.
• Identification of a restriction enzyme allowing allele
discrimination by in-silico analysis can be done using the
program designated NEBcutter V2, which has an option for
viewing fragments of an in-silico digest .
PCR Reagents : ( Total reaction vol 50ul )
1. Taq DNA polymerase (0.5ul)
2. dNTPs (1ul; 10mM)
3. Foreward & reverse primers (1ul)
4. 2ul genomic DNA template

6.
5. 10 x Taq Buffer ( standard Taq containing Mg+2 )
6. 39.5 ul Distilled water
Procedure :
1. Label three 0.5ml microfuge tube RSM, 1 & 2. Place tubes on
ice.
2. Prepare reaction stock mixture for three PCR reactions to account
for pipetting loses. First, calculate the amount of water necessary
for the desired volume and add it to the tube. Then add buffer &
the remaining compounds. Add enzyme last and mix by pipetting
up & down several times. Never mix by voetexing because Taq
polymerase is very sensitive to vortexing.
3. Place tube into the thermal cycler & amplify DNA using standard
conditions. Typically these are : initial denaturation at 95°C for 3
minutes followed by 30 to 35 cycles consisting of denaturation
(D) at 94°C for 30 seconds, Annealing ( A ) at 55 - 60°C for 30
seconds, Elongation ( E ) at 72°C for 30 2 minutes.
4. Set annealing temperature 5°C below the primer melting
temperature (Tm) & Tm of both primers of forward and reverse
should be closed or same.
5. Final extension for 5 minuted at 74°C .
6. Run 2ul DNA on a gel to check size & concentration of PCR
products .

7.
PCR – process
# Restriction digestion of PCR amplified product :
Restriction Digestion—Reactions contained 2ul of amplified DNA
and 1ul of restriction enzyme in appropriate buffer at a volume of 20ul
total. Digestions were incubated 45–60 min at 37 °C and stored at -20
°C until use.
# Gel electrophoresis of digested PCR amplified product :
Principle :Gel electrophoresis is a laboratory technique used to
separate charged molecules. DNA is negatively charged and moves,
under the force of an electric current, throught the matrix of agarose
gel. Molecules separate by size, with the smaller ones moving more
rapidly through the gel than the large ones. The rate of migration is
proportional to size, smaller fragments move more quickly, and wind
up at the bottom at the gel.

8.
Reagents :
1. Agarose (1%)
2. 1 x TAE buffer -100mM
3. 6 x gel loading dye
4. EtBr (5ug/ml)
5. Ladder ( 1 -5 kb)
Making an Agarose Gel :
1. Measure 1 g of agarose.
2. Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
3. Microwave for 1-3 min until the agarose is completely dissolved.
4. Let agarose solution cool down to about 50°C (about when you can
comfortably keep your hand on the flask), about 5 mins.
5. (Optional) Add ethidium bromide (EtBr) to a final concentration of
approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock
solution per 100 mL gel). EtBr binds to the DNA and allows you to
visualize the DNA under ultraviolet (UV) light.
6. Pour the agarose into a gel tray with the well comb in place.
7. Place newly poured gel at 4°C for 10-15 mins OR let sit at room
temperature for 20-30 mins, until it has completely solidified.

9.
Gel agarose making & pouring gel into gel casting tray
DNA loading into gel
Loading Samples and Running an Agarose Gel:
1. Add loading buffer to each of your digest samples.
2. Once solidified, place the agarose gel into the gel box
(electrophoresis unit).
3. Fill gel box with 1xTAE (or TBE) until the gel is covered.
4. Carefully load a molecular weight ladder into the first lane of the
gel.

10.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 80-150 V until the dye line is approximately 75-
80% of the way down the gel.
7. Turn OFF power, disconnect the electrodes from the power
source, and then carefully remove the gel from the gel box.
8. (Optional) If you did not add EtBr to the gel and buffer, place
the gel into a container filled with 100 mL of TAE running
buffer and 5 μL of EtBr, place on a rocker for 20-30 mins,
replace EtBr solution with water and destain for 5 mins.
9. Remove the gel from the casting tray and visualize under UV
transluminator & photographs is taken by gel doc. Computer system. #
Visualize the digested DNA fragments

11.
Observation :
Different size of bands show that DNA sample have restriction
site for particular restriction endo-nuclease . Lengths of digested DNA
fragment is compared with standard ladder DNA sample.
Results :
Different size of bands are observed in gel by Gel DOC picture.
Precautions :
1. Safety precautions included handling all ethidium bromide
(EtBr) solutions and gels with disposable gloves and wearing
ultraviolet-protective goggles when viewing gels on the
transilluminator. EtBr was removed from contaminated solutions
by filtration prior to disposal.
2. DNA sample should not be sheared & contaminated before
using for PCR amplification.
3. All instruments should be cleaned & sterilized.
4. Primers length should not be more than 18-25 bases.
5. If your target DNA sequence is GC-rich, increase the time of the
denaturing.
6. Your 5’ and 3’ primers should be designed to have similar
melting temperatures (Tm).
7. Set the annealing temperature to 5°C lower than the Tm of your
primers. If you are getting non-specific PCR products, increase
the annealing temperature step-wise by 1-2°C.
***