CERTIFICATE OF ANALYSIS

For

Version 1 (110804)

Catalog Size i-Taq6 500 units

For Research Use Only and Not For Human and Animal Therapeutic and Diagnostic Use. Disclaimer:

THE PRODUCTS FROM I-DNA ARE PROVIDED AS-IS WITH NO WARRANTY EITHER EXPRESS OR IMPLIED, AND SPECIFICALLY WITHOUT ANY WARRANTY OF SUCCESS, REPRODUCIBILITY OR FITNESS FOR A PARTICULAR PURPOSE OR SCIENTIFIC APPLICATION, MERCHANTABILITY OR NON-INFRINGEMENT. I-DNA ASSUMES NO RESPONSIBILITY WHATSOEVER WITH RESPECT OF THE USE, SALE, OR DISPOSITION BY PURCHASER OF PRODUCTS. PURCHASER SHALL INDEMNIFY AND HOLD I-DNA HARMLESS FROM AND AGAINST ANY AND ALL CLAIMS, DAMAGES, COSTS, EXPENSES AND OTHER 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 Fax: 6776 0368 Email: sales@i-dna.sg

Red i-Taq DNA Polymerase

Red 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. An inert red dye is added to i-Taq DNA polymerase (cat# i-Taq5) to produce a new product called Red i-Taq DNA polymerase (cat# i-Taq6). The red dye allows visual monitoring of the electrophoresis and identification of the reaction containing the enzyme. The red dye has no adverse effect on reaction. In addition, Red i-Taq DNA polymerase is supplied as 1 unit/µl, making it more convenient to use. Materials Provided: Red i-Taq DNA polymerase is supplied with

• i-Taq DNA polymerase, 1 units/µl. Size = 500 units. Volume = 500 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 Size i-Taq6 500 units Storage: -20oC (upon receiving and is stable for 24 months from this date)

Lot Number: refer to label on the product vial.

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 i-

Taq 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 Volume (µl) Final concentration 10X Assay Buffer C 5 1X 50 mM MgCl2 solution 1.5 a 1.5 mM a 10 mM dNTP mastermix 1 200 µM Forward primer variable 0.2 – 1 µM Reverse primer variable 0.2 – 1 µM Red i-Taq DNA Polymerase (1 u/µl) 1 – 2.5 1 – 2.5 unit Sterile H2O Adjust to 40 µl

Working Amplification Mastermix 40 µl

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 Add 50 mM MgCl2 solution

1.0 mM 1.5 mM

1.0 µl 1.5 µl

1.75 mM 1.75 µl 2.0 mM 2.0 µl 2.5 mM 2.5 µl 3.0 mM 3.0 µl 4.0 mM 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 Volume (µl) Final concentration Working Amplification Mastermix 40 1X Template DNA 10 < 1 µg

Total Volume of Reaction 50 µl 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 Temp. (oC) Time Cycle Initial Denaturation 94 5 min 1 Denaturation 94 30 sec

25 – 35 c Annealing 50 – 65 b 45 sec Extension 72 30 sec / 1kb c Final Extension 72 1 min 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.