DNA Amplification GeNeiTM DNA Amplification GeNeiTM

DNA Amplification
Reagent Kit
Manual
(with Marker)

0660300051730
Cat No.
0660500011730
Revision No.0004017

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

CONTENTS

Page No.

v Introduction 1
v Description 2
v Materials Provided 7-8
v Procedure 11
v Trouble Shooting 17
v References 21
v Ordering Information 22
v Other Related Products 23
v Flow Chart 25-27

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DNA Amplification GeNeiTM DNA Amplification GeNei

Introduction: l dNTPs or nucleotides which get added to form

Invented in 1983 by Kary Mullis, the Polymerase Chain the new strand - the DNA building blocks
Reaction or PCR is a reliable and routinely used tool l Buffers including divalent and monovalent cations
in Molecular Biology and Biotechnology. The purpose to support the reaction.
of PCR is to rapidly amplify and make a large number
of copies of any gene starting from a very small Description:
number. Kary Mullis was awarded the Nobel Prize in This kit is based on a very simple method for the
Chemistry for his work in 1993. The reaction is very in-vitro amplification of specific nucleic acids using
simple and requires no more than a test tube, a few Taq DNA polymerase and minimum two
simple reagents and a source of heat. oligonucleotides specific to the DNA to be amplified.
The reaction mixture comprises of the Briefly, the technique is carried out as follows:
following:
l A target or template dsDNA which is the DNA
1. Initiation step: The first step sometimes involves
that is to be amplified heating the reaction to a temperature of 94-96°C
l Two short and specific primers which are
just for short while to activate the enzyme used.
responsible for initiation of amplification called Denaturation: Involves heating the reaction
forward and reverse primers. They contain mixture to a temperature of 94 - 96°C at which
sequences complementary to the target DNA and the double stranded DNA of the template
are single stranded undergoes degradation by breaking of hydrogen
l A thermostable DNA polymerase which brings
bonds which hold the two strands together,
about the polymerization of the dNTPs thereby forming single stranded DNA.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

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

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Fig: Schematic representation of steps Highlights:

involved in DNA amplification by PCR: • Quick and reliable method
• Useful for in-vitro amplification of DNA isolated
from any source
• Contains all necessary reagents
• 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 has
also become an attractive technique used in
diagnostics and the Diagnostic Industry.
Time taken: 1 hour

1. Denaturation 3. Extension
2. Anealing 4. Repeat cycles
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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Materials Provided: Materials Provided:

The list below provides information about the The list below provides information about the
materials supplied in the kit. The components should materials supplied in the kit. The components should
be stored as suggested. be stored as suggested.
Quantity Quantity
Materials 0660300051730 Store Materials 0660500011730 Store
(100 Reactions) (50 Reactions)
Taq DNA Polymerase 84 µl -20°C Taq DNA Polymerase 42 µl -20°C
(3 U/µl) (3 U/µl)
10X Taq DNA Polymerase 1.2 ml -20°C 10X Taq DNA Polymerase 0.6 ml -20°C
Buffer with MgCl2 Buffer with MgCl2
10X Taq DNA Polymerase 0.6 ml -20°C 10X Taq DNA Polymerase 0.25 ml -20°C
Buffer without MgCl2 Buffer without MgCl 2
25 mM MgCl2 0.25 ml -20°C 25 mM MgCl 2 0.1 ml -20°C
10 mM dNTP Mix 0.4 ml -20°C 10 mM dNTP Mix 0.2 ml -20°C
StepUp™100 bp DNA 100 µl -20°C StepUp™100 bp DNA 50 µl -20°C
Ladder (Ready To Use) Ladder (Ready To Use)
Control DNA Template 20 µl -20°C Control DNA Template 10 µl -20°C
Control Forward Primer 20 µl -20°C Control Forward Primer 10 µl -20°C
Control Reverse Primer 20 µl -20°C Control Reverse Primer 10 µl -20°C
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DNA Amplification GeNeiTM DNA Amplification GeNei

Materials Required but not provided: Notes:

Equipment : Thermocycler, Dry Bath/ Water Bath. • Read the entire procedure carefully
prior to starting the experiment
Reagents : Sterile Milli Q or Autoclaved double
• It is ideal to perform amplification reaction
distilled water
in a dedicated room using reagents and
Other Requirements : 0.5/ 0.2 /1.5/2 ml equipment also dedicated for amplification
Microcentrifuge tubes, reaction.
Micropipettes, Gloves, • All concentrated stocks of target
Micro Tips preferably sequences should be kept away from
with filter barrier and amplification area and equipment.
Mineral Oil. • Meticulous care must be taken to avoid
carrryover of DNA from one tube to another
in order to prevent false positives.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Procedure: 2. Mix the solution gently.

1. To a sterile 0.2 / 0.5 ml microfuge tube, add the 3. Layer the reaction mix with 50 µl of mineral oil to
reagents in the order as indicated in table below: avoid any evaporation (Optional).
(For Control Reaction) Note: Mineral oil need not be added if the
Thermocycler is equipped with a heated
Reagent Amount lid.
Sterile MilliQ water or Autoclaved 38 µl PCR Amplification
double distilled water
4. Carry out the amplification in a thermocycler for
10X Taq Polymerase Assay buffer 5 µl 30 cycles using the following reaction
with MgCl2 conditions:
10 mM dNTP mix (2.5 mM each) 3 µl (For Control Reaction only)
Initial Final
Control Template DNA 1 µl Denaturation Denaturation Annealing Extension Extension
Control Forward Primer 1 µl 94ºC 94°C 48°C 72°C 72°C
Control Reverse Primer 1 µl 1 minute 30 sec. 30 sec. 1 min. 2 min.
Taq DNA Polymerase 1-2 U
Note: In case of Test reaction, carry out
amplification as per optimized PCR conditions.
Note: In case of Test reaction, use Assay
Buffer with MgCl2 . To Assay Buffer without
MgCl2, add optimized amount of MgCl2.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Analysis on Agarose Gel Template DNA: Typically nanogram amounts of

5. After the reaction is completed, take out the cloned template, up to microgram amount of genomic
reaction mix and electrophorese 10 µl of the DNA or 20,000 target copies are chosen to start
aqueous layer along with StepUp™ 100 bp DNA optimization trials. Higher amounts of the template
ladder on 1% agarose gel for 1 to 2 hours at 100 DNA inhibits or results in non-specific amplifications.
volts.
Primers: Primers are oligonucleotides, typically 15
6. Visualize under UV light for 0.8 Kb DNA fragment. to 30 bases long. It is important that the primers do
Some Important Tips: not contain more than two bases complimentary with
For the efficient amplification of target sequences each other, especially at their 3' ends to avoid the
individual reaction components, time and temperature formation of PRIMER DIMER. There should not be
parameters must be adjusted. The following any internal secondary structure. Typically, 40 to
suggestions may help to carry out gene amplifications 60% G+C content is often recommended. Optimal
successfully. annealing temperature must be determined
empirically. The concentration of the primers typically
Sample volume and microfuge tube: Most should be within 0.1 to 1 µM range. Calculated Tm
amplifications are performed at 20, 50 or 100 µl volume for both primers used in reaction should not differ
in 0.2 or 0.5 ml microfuge tubes. Larger volumes are >5°C, and Tm of the amplification product should not
not recommended as there will not be adequate differ from primers by >10°C. Annealing temperature
thermal equilibrium of the reaction mixture. usually is 5°C below the calculated lower Tm.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Deoxynucleotidetriphosphates: Typically, the Thermal profiles: DNA denaturation is the critical

final concentration of each dNTP in a standard step in the DNA amplification reactions. The time
amplification reaction mixture is 200 mM. It is important specified for DNA depends on various factors like,
to keep the four dNTP concentrations above the nature of template, GC content, secondary structure
estimated Km of each dNTP (10 to 15 mM) and etc., For most amplification, incubation time for DNA
balanced for best base incorporation fidelity. denaturation is 30 seconds - 1 minute at 92 to 95°C,
with 94°C being standard choice.
Taq DNA Polymerase Buffer: It is advisable to
carry out a titration series in small increments over
Primer annealing in most of the cases is done 37 to
the 1.5 to 4 mM range to locate the magnesium 55°C. The specified annealing temperature is
concentration producing the highest yield of a specific
calculated empirically and is usually 5°C less than
product. Too little free Magnesium will result in no the melting temperature (Tm) of the primers used.
amplification and too much may produce a variety of
Extreme annealing temperature may result in non-
unwanted products. specific amplification.
Taq DNA Polymerase: Taq DNA polymerase is a 94 Primer extension, in most amplifications occurs
kD thermostable DNA polymerase which lacks 3' to
efficiently at a temperature of 72°C and seldom
5' exonuclease activity but has 5' to 3' exonuclease needs optimization. Time specified for extension
activity in addition to 5' to 3' polymerase activity. For
depends on the length of target sequence. For
most amplification reactions 1.5 to 2 units of enzyme example for 1 kb target DNA usually 1 minute
are recommended as excess of it leads to non-
extension is recommended.
specific amplification.The enzyme can incorporate
base analogs such as 7-deaza- dGTP very efficiently.

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DNA Amplification GeNeiTM DNA Amplification GeNei

Trouble Shooting:
Observation: There is no yield of the desired Possible Suggestions
product cause
Possible Suggestions Proteases or Examine need to increase
cause nucleases in preincubation at 95°C for 5 to 10
the sample. minutes in the absence of enzyme
Enzyme not Make sure that the enzyme is to inactivate harmful proteases or
added being added reproducibly, nucleases in the sample.
appropriately preferably in a master mix.
Contamination There could be excessive
Incomplete l Ensure complete denaturation contaminants carried over in the
in the sample
denaturation of DNA in each cycle by using sample. Dilute the sample and
properly sized 0.2 / 0.5 ml repeat PCR.
microfuge tubes and by
allowing sufficient time at the Enzyme The enzyme could be inactive if
denaturation plateau inactivation not stored properly. Ensure
temperature. proper storage of enzyme.
l Consider a slightly higher
denaturation temperature.

Primer condition Check the chemical integrity of

the primers.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Observation: A smear or non specific amplified Observation: A primer-dimer artifact band is seen
product is seen on the agarose gel. on the agarose gel
Possible Suggestions Possible Suggestions
cause cause
Excess Reduce Taq DNA polymerase Primer check primer sequences for 3'
Enzyme concentration sequence may complimentarity
is used not be
Annealing complimentary
l Increase annealing
temperature temperature Incorrect Design longer primers
may not l Minimize the incubation times Primer Design
be correct of the annealing and
High Primer Reduce primer concentration
extension steps
concentration
Number of Decrease the number of cycles
Cycles Amount of Increase amount of target DNA
may be to DNA
many insufficient

Product size Always load Molecular Weight

size markers to determine the size
of the amplified product.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

References: Ordering Information

1. Saiki R.K et.al. (1985) Science 230, 1350-1354. Product Size Cat #
2. Mullis K.B et.al. (1987) Methods Enzymol. 155, DNA Amplification 1 EA 0660300051730
335-350 Reagent Kit (With Marker)
3. Saiki R.K et.al. (1988) Science 239, 487-491. (Includes consumables
4. PCR Technology, H. Erlich Ed., Stockton press, for 100 Reactions)
New York, 1989.
5. PCR Topics, A. Rolfs, H.C. Schumacher, P. Marx DNA Amplification 1 EA 0660500011730
Eds. Springer-Verlag, New York, 1991. Reagent Kit (With Marker)
6. PCR Protocols, Current methods and applications (Includes consumables
Ed. Bruce A. White, Volume 15, Humana press, for 50 Reactions)
Totowa, New Jersey,1993.

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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

Other Related Products:

Product Cat #
610667400011730 M-MuLV RT-PCR Kit
610661600021730 One Step AMV RT-PCR Kit
610661700021730 One Step M-MuLV RT-PCR Kit
610692470071730 Random Hexamer (6 mer)
610690970071730 Oligo dT primer pd (T)18
612602300011730 Nucleic acid agarose gel
Electrophoresis Kit
612107180051730 PCR Klenzol
612115300031730 PCR Purification Kit

Email:
Sales: [email protected]
Customer Support:
[email protected]

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DNA Amplification GeNeiTM DNA Amplification GeNei
Flow Chart:
For Control Reaction
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 Control Template DNA
•

P.T.O
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DNA Amplification GeNeiTM DNA Amplification GeNeiTM

•
Add 1 µl each of Control Forward
and Reverse Primers
•
Add 1-2 U of Taq DNA Polymerase

•
Mix and Layer with 50 µl of Mineral Oil
•
PCR conditions (For Control Reaction)
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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