DNA Amplification GeNeiTM DNA Amplification GeNeiTM

DNA Amplification
Core Kit Manual
(without Marker)

Cat No. 0660400051730

Revision No.: 00050516

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

CONTENTS

Page No.

v Introduction 1

v Description 1

v Materials Provided 8

v Procedure 9

v Trouble Shooting 13

v References 15

v Ordering Information 16

v Other Related Products 16

v Flow Chart 18

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Introduction:
Invented in 1983 by Kary Mullis, the Polymerase Chain
Reaction or PCR is a reliable and routinely used tool in
Molecular Biology and Biotechnology. The purpose of PCR
is to rapidly amplify and make a large number of copies of
any gene starting from a very small number. Kary Mullis was
awarded the Nobel Prize in Chemistry for his work in 1993.
The reaction is very simple and requires no more than a test
tube, a few simple reagents and a source of heat.
The reaction mixture comprises of the following:
l A target or template dsDNA which is the DNA that is to
be amplified
l Two short and specific primers which are responsible for
initiation of amplification called forward and reverse
primers. They contain sequences complementary to the
target DNA and are single stranded
l A thermostable DNA polymerase which brings about the
polymerization of the dNTPs
l dNTPs or nucleotides which get added to form the new
strand - the DNA building blocks
l Buffers including divalent and monovalent cations to
support the reaction.

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Description: 4. Final extension: This single step is occasionally

This kit is based on a very simple method for the in vitro performed at a temperature of 70-74°C for 5-15 minutes
amplification of specific nucleic acids using Taq DNA after the last PCR cycle to ensure that any remaining
polymerase and minimum two oligonucleotides specific to single-stranded DNA is fully extended.
the DNA to be amplified. After this the entire process or cycle is repeated with the
Briefly, the technique is carried out as follows: newly formed DNA acting as target DNA. Repetition of these
cycles results in the amplification of target DNA generating
1. Initiation step: The first step sometimes involves heating millions of copies in a very short time.
the reaction to a temperature of 94-96°C just for short
while to activate the enzyme used. Denaturation: Involves This kit recommends 30 cycles.
heating the reaction mixture to a temperature of 94 - 96°C The entire reaction is carried out in a thermocycler.
at which the double stranded DNA of the template
undergoes degradation by breaking of hydrogen bonds In addition to the products a standard DNA preparation or
which hold the two strands together, thereby forming single ladder of DNA fragments of known size is also run because
stranded DNA. of which the size of PCR products can be determined.
2. Annealing: The next step involves cooling the reaction
mixture to 54°C at which annealing takes place. In this 5. Final hold: This step at 4-15 °C for an indefinite time
step the primers bind to the separated strands of template may be employed for short-term storage of the reaction
DNA due to their complementarity with target DNA.
3. Extension: In the next step the polymerase binds to this
dsDNA formed by linking between the primer and template
and the temperature is maintained at 74°C. At this
temperature, the polymerase binds and "reads" the
template and adds complementary bases as it moves
along the template in the 5' - 3' direction, condensing the
5'-phosphate group of the dNTPs with the 3'-hydroxyl group
at the end of the nascent (extending) DNA strand. This
results in the synthesis of a new DNA strand. The
polymerase also can proof read so as to correct any wrong
base that it has added. The polymerase used is a special
polymerase capable of withstanding high temperatures.
It is isolated from the bacterium Thermus aquaticus and
its optimum activity temperature is around 75°C.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Fig: Schematic representation of steps involved in DNA Highlights:

amplification by PCR: l Quick and reliable method
l Useful for in-vitro amplification of DNA isolated from any
source
l Contains all necessary reagents
l Highly sensitive and specific

Application:
This seemingly simple technique finds wide applications in
Life Science Research in the detection of hereditary diseases,
identification of genetic fingerprints, diagnosis of infectious
diseases, cloning of genes, paternity testing and DNA
computing among others. Since the advent of the technology,
PCR soon has also become an attractive technique used in
diagnostics and the Diagnostic Industry.
Time taken: 1 hour

1. Denaturation
2. Anealing
3. Extension
4. Repeat cycles

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Materials Provided: Note:

The list below provides information about the materials • Read the entire procedure before starting the
supplied in the kit. The components should be stored as experiment.
suggested. • It is ideal to perform amplification reaction in a dedicated
Quantity room using reagents and equipment also dedicated for
amplification reaction.
Materials 0660400051730 Store
• All concentrated stocks of target sequences should
(100 Reactions)
be kept away from amplification area and equipment.
Taq DNA Polymerase 84 µl -20°C • Meticulous care must be taken to avoid carrryover of
(3 U/µl) DNA from one tube to another in order to prevent false
10X Taq DNA 1.2 ml -20°C
positives.
Polymerase Buffer
with MgCl2 Procedure:
10X Taq DNA 0.6 ml -20°C 1. To a sterile 0.2 / 0.5 ml microfuge tube, add the reagents
Polymerase Buffer in the order as indicated in table below:
without MgCl2
Reagent Amount
25 mM MgCl2 0.25 ml -20°C Sterile MilliQ water or Autoclaved 38 µl
double distilled water
10 mM dNTP Mix 0.4 ml -20°C
10X Taq Polymerase Assay buffer 5 µl
StepUp™ 100 bp DNA with MgCl2
Ladder (Ready To Use) 100 µl -20°C
10 mM dNTP mix (2.5 mM each) 3 µl
Taq DNA Polymerase 1-2 U
Materials Required but not provided:
Equipment : Thermocycler, Dry Baths/ Water Bath.
Reagents : Sterile Milli Q or Autoclaved double
distilled water
Other Requirements : 0.5/ 0.2 /1.5/2 ml Microcentrifuge
tubes, Micropipettes, Gloves,
Micro Tips preferably with filter
barrier and Mineral Oil.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

2. Mix the solution gently. Template DNA: Typically nanogram amounts of cloned
3. Layer the reaction mix with 50 µl of mineral oil to avoid template, up to microgram amount of genomic DNA or 20,000
any evaporation (Optional). target copies are chosen to start optimization trials. Higher
Note: Mineral oil need not be added if the amounts of the template DNA inhibits or results in non-
Thermocycler is equipped with a heated lid. specific amplifications.
PCR Amplification Primers: Primers are oligonucleotides, typically 15 to 30
bases long. It is important that the primers do not contain
4. Carry out the amplification in a thermocycler for 30 cycles
more than two bases complimentary with each other,
using the following reaction conditions:
especially at their 3' ends to avoid the formation of PRIMER
Initial Final DIMER. There should not be any internal secondary structure.
Denaturation Denaturation Annealing Extension Extension Typically, 40 to 60% G+C content is often recommended.
94ºC 94°C 48°C 72°C 72°C Optimal annealing temperature must be determined
1 minute 30 sec. 30 sec. 1 min. 2 min.
empirically. The concentration of the primers typically should
be within 0.1 to 1 µM range. Calculated Tm for both primers
Analysis on Agarose Gel
used in reaction should not differ >5°C, and Tm of the
5. After the reaction is completed, take out the reaction mix
amplification product should not differ from primers by >10°C.
and electrophorese 10 µl of the aqueous layer along with
Annealing temperature usually is 5°C below the calculated
StepUp™ 100 bp DNA ladder on 1% agarose gel for 1 to
lower Tm.
2 hours at 100 volts.
Deoxynucleotidetriphosphates: Typically, the final
6. Visualize under UV light.
concentration of each dNTP in a standard amplification
Some Important Tips: reaction mixture is 200 mM. It is important to keep the four
For the efficient amplification of target sequences individual dNTP concentrations above the estimated Km of each dNTP
reaction components, time and temperature parameters must (10 to 15 mM) and balanced for best base incorporation fidelity.
be adjusted. The following suggestions may help to carry
out gene amplifications successfully.
Sample volume and microfuge tube: Most amplifications
are performed at 20, 50 or 100 µl volume in 0.2 or 0.5 ml
microfuge tubes. Larger volumes are not recommended as
there will not be adequate thermal equilibrium of the reaction
mixture.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Taq DNA Polymerase Buffer: It is advisable to carry out a Trouble Shooting:

titration series in small increments over the 1.5 to 4 mM Observation: There is no yield of the desired product
range to locate the magnesium concentration producing the
highest yield of a specific product. Too little free Magnesium Possible Cause Suggestions
will result in no amplification and too much may produce a
variety of unwanted products. Enzyme not added Make sure that the enzyme is being
appropriately added reproducibly, preferably in a
Taq DNA Polymerase: Taq DNA polymerase is a 94 kD master mix.
thermostable DNA polymerase which lacks 3' to 5' Ensure complete denaturation of
Incomplete l
exonuclease activity but has 5' to 3' exonuclease activity in DNA in each cycle by using
denaturation 2 2
addition to 5' to 3' polymerase activity. For most amplification properly sized 0.2 / 0.5 ml
reactions 1.5 to 2 units of enzyme are recommended as
microfuge tubes and by allowing 3
excess of it leads to non-specific amplification.The enzyme sufficient time at the denaturation
can incorporate base analogs such as 7-deaza- dGTP very plateau temperature.
efficiently.
l Consider a slightly higher
Thermal profiles: DNA denaturation is the critical step in denaturation temperature.
the DNA amplification reactions. The time specified for DNA Primer condition Check the chemical integrity of the
depends on various factors like, nature of template, GC
primers.
content, secondary structure etc., For most amplification,
incubation time for DNA denaturation is 30 seconds - 1 minute Proteases or Examine need to increase
at 92 to 95°C, with 94°C being standard choice. nucleases in the preincubation at 95°C for 5 to 10
sample. minutes in the absence of enzyme
Primer annealing in most of the cases is done 37 to 55°C. to inactivate harmful proteases or
The specified annealing temperature is calculated empirically nucleases in the sample.
and is usually 5°C less than the melting temperature (Tm) of
the primers used. Extreme annealing temperature may result Contamination in the There could be excessive
in non-specific amplification. sample contaminants carried over in the
sample. Dilute the sample and
Primer extension, in most amplifications occurs efficiently repeat PCR.
at a temperature of 72°C and seldom needs optimization.
Time specified for extension depends on the length of target Enzyme inactivation The enzyme could be inactive if not
sequence. For example for 1 kb target DNA usually 1 minute stored properly. Ensure proper
extension is recommended. storage of enzyme.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Observation: A smear or non specific amplified product is References:

seen on the agarose gel. 1. Saiki R.K et.al. (1985) Science 230, 1350-1354.
2. Mullis K.B et.al. (1987) Methods Enzymol. 155, 335-350
Possible Cause Suggestions
3. Saiki R.K et.al. (1988) Science 239, 487-491.
Excess Enzyme Reduce Taq DNA polymerase 4. PCR Technology, H. Erlich Ed., stockton press, New York,
is used concentration 1989.
5. PCR Topics, A. Rolfs, H.C. Schumacher, P. Marx Eds.
Annealing l Increase annealing temperature Springer-Verlag, New York, 1991.
temperature may
2 not l Minimize the incubation2 times of 6. PCR Protocols, Current methods and applications Ed.
be correct the annealing and extension Bruce A. White, Volume 15, Humana press, Totowa, New
steps 3 Jersey,1993.

Number of Cycles Decrease the number of cycles

may be to many
Product size Always load Molecular Weight size
markers to determine the size of the
amplified product.

Observation: A primer-dimer artifact band is seen on the

agarose gel

Possible Cause Suggestions

Primer sequence check primer sequences for 3'

may not be complimentarity
complimentary
Incorrect Primer Design longer primers
Design
High Primer Reduce primer concentration
concentration
Amount of DNA Increase amount of target DNA
insufficient

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Ordering Information

Product Size Cat #

DNA Amplification Core Kit 1 EA 0660400051730

(Without Marker)
(Includes consumables
for 100 Reactions)

Other related products:

Cat # Product
0667400011730 M-MuLV RT-PCR Kit
0661600021730 One Step AMV RT-PCR Kit
0661700021730 One Step M-MuLV RT-PCR Kit
0692470071730 Random Hexamer (6 mer)
0690970071730 Oligo dT primer pd (T)18
2602300011730 Nucleic acid agarose gel
Electrophoresis Kit
2107180051730 PCR Klenzol
2115300031730 PCR Purification Kit

Email:
Sales:
sales@geneilabs.com

Customer Support:
techsupport@geneilabs.com

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Flow Chart

Take 38 µl of MilliQ water or autoclaved double

distilled in 0.2 or 0.5 ml Microcentrifuge tube

Add 5 µl of 10X Taq Polymerase Assay Buffer

with Magnesium Chloride

Add 3 µl of 10 mM dNTP Mix

Add 1 µl of Template DNA

Add 1 µl each of Forward and Reverse Primers

Add 1-2 U of Taq DNA Polymerase

Mix and Layer with 50 µl of Mineral Oil

PCR conditions
l Initiation at 94°C - 1 min
l Denaturing: 94°C, 30 secs

l Annealing: 48°C: 30 secs

l Extension: 72°C: 1 min

30 cycles

Final extension at 72°C for 2 or more minutes. Analyze

10 µl of PCR product along with StepUp™ 100 bp DNA
Ladder by Agarose Gel Electrophoresis.

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