CERTIFICATE OF ANALYSIS
For
i-Taq DNA Polymerase
Version 1 (110804)
Catalog
i-Taq5
Size
500 units
For Research Use Only and Not For Human and Animal Therapeutic and Diagnostic Use.
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LIABILITIES WITH RESPECT TO PURCHASER’S USE OF THE PRODUCT.
For sales and technical support, please contact i-DNA Biotechnology Pte Ltd at:
Tel: 6779 0665
1
Fax: 6776 0368
Email: sales@i-dna.sg
i-Taq DNA Polymerase
Product Description: i-Taq DNA polymerase is a purified recombinant thermostable
DNA polymerase enzyme, which has 5’-3’ polymerase activity with optimum activity at
74oC. The enzyme exhibits high thermal stability in prolonged incubation at 95°C. Note:
this i-Taq DNA polymerase lacks 3’-5’ exonuclease activity.
Materials Provided: i-Taq DNA polymerase is supplied with

i-Taq DNA polymerase, 5 units/µl. Size = 500 units. Volume = 100 µl.
Notes:
o one unit is defined as the amount of enzyme required to catalyze the
incorporation of 10nmol of dNTP into acid-insoluble form in 30 minutes at
74°C in the presence of the reaction buffer
o i-Taq DNA polymerase is stored in 20 mM Tris-HCl (pH 8.0 at 25 °C), 30
mM KCl, 0.1 mM DTT, 0.5% Tween 20, 50% (v/v) glycerol.

10X Assay Buffer C, containing no MgCl2, consists of 670 mM Tris-HCl (pH 8.8 at
25°C), 166 mM (NH4)2SO4, 4.5% Triton®-X-100, 2 mg/ml gelatin. Volume = 1
ml.
Notes:
o Add MgCl2 to 10X Assay Buffer C accordingly as the final MgCl2
concentration required may vary from reaction to reaction.
o i-Taq DNA polymerase (cat# i-Taq5) is optimized with 10X Assay Buffer C.

50mM MgCl2 solution. Volume = 0.5 ml.
Catalog
i-Taq5
Size
500 units
Storage:
-20oC (upon receiving and is stable for 24 months from this date)
Lot Number:
refer to label on the product vial.
2
Quality Control:
1. Absence of exonuclease activity: 1µg of λDNA/Hind III digest incubated with 10 units
of i-Taq DNA polymerase at 70oC for 4 hrs showed no alteration in banding pattern.
2. Absence of endonuclease activity: 1µg of λDNA incubated with 10 units of i-Taq DNA
polymerase at 70oC for 4 hrs showed no degradation of λDNA.
3. Absence of nicking activity: 1µg supercoiled pBR322 DNA incubated with 10 units of iTaq DNA polymerase at 70oC for 4 hrs showed no relaxation of supercoiled DNA.
4. Ribonuclease activity: No detectable degradation of 28S/18S bands was observed after
incubation of 10 units of Taq DNA polymerase with 1 µg of total RNA for 4 hours at
70°C
5. Functional activity: 0.1 ng of lambda DNA was amplified using specific primers to
produce a distinct 500 bp band.
6. Extreme Thermostability: i-Taq DNA polymerase is highly active at temperatures
around 74oC and it does not lose its activity considerably even after prolonged
incubation at high temperature.
Recommended Protocol
Before starting a new reaction, it is important to be aware that the optimal reaction
condition (such as incubation temperature and time, concentration of template DNA,
concentration of primer, concentration of magnesium ions) depends on template and primer
pair used. For GC-rich template or amplicon region, this Taq DNA polymerase may not be
suitable for this specific application. The recommended protocol below is general and is
not specific for any template or primer. Its purpose is to provide a recommended range for
each component in the reaction for the optimization. It is strongly recommended that the
end-user optimizes the reaction condition for new template and primer pair.
Step 1 - Prepare the Working Amplification Mastermix by adding the following
components on ice:
Component
10X Assay Buffer C
50 mM MgCl2 solution
10 mM dNTP mastermix
Forward primer
Reverse primer
i-Taq DNA Polymerase (5 u/µl)
Sterile H2O
Working Amplification Mastermix
3
Volume (µl)
5
1.5 a
1
variable
variable
0.2 – 0.5
Adjust to 40 µl
40 µl
Final concentration
1X
1.5 mM a
200 µM
0.2 – 1 µM
0.2 – 1 µM
1 – 2.5 unit
Notes:
Scale up the Working Amplification Mastermix accordingly, depending on the
number of reactions required. Prepare 10% overage for easy pipetting purpose.
a
The final concentration of MgCl2 varies from reaction to reaction. For a new
reaction, it is recommended to optimize the final concentration of MgCl2 first
between 1 – 4 mM range. Refer to the table below:
Final concentration of MgCl2 in 50 µl
reaction volume
1.0 mM
1.5 mM
1.75 mM
2.0 mM
2.5 mM
3.0 mM
4.0 mM
Add 50 mM MgCl2 solution
1.0 µl
1.5 µl
1.75 µl
2.0 µl
2.5 µl
3.0 µl
4.0 µl
Step 2 – Into a reaction tube, reaction strip or reaction plate, combine Working
Amplification Mastermix and template DNA as shown below.
Component
Working Amplification Mastermix
Template DNA
Total Volume of Reaction
Volume (µl)
40
10
50 µl
Final concentration
1X
< 1 µg
Note: DO NOT use more than 1 µg of template DNA as this may produce variable results.
Recommended Thermal Cycling Program
Regardless of the thermal cycler brand or model, set the thermal cycling program as shown
below. To avoid evaporation of the reaction mix during thermal cycling, use mineral oil or
heated lid.
Step
Initial Denaturation
Denaturation
Annealing
Extension
Final Extension
Temp. (oC)
94
94
50 – 65 b
72
72
Time
5 min
30 sec
45 sec
30 sec / 1kb
1 min
Cycle
1
25 – 35
c
c
1
Notes:
b
Annealing temperature varies from primer to primer. Most primer works well
within the annealing temperature range 50 – 65 oC. Alternatively, determine the
annealing temperature by taking approximately 5 °C below the melting temperature
of primer.
c
We recommend 25 – 35 cycles.
d
For extension time, use 30 sec for every 1kb of amplified amplicon. For example,
if the amplicon is 500 bp, use 30 sec. If the amplicon is 1.8kb, use 60 sec.
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