Bio-Rad iProof High-Fidelity DNA Polymerase User Manual

iProof™High-Fidelity DNA Polymerase

2 units/µl, 10 µl 20U 172-5300

2 units/µl, 50 µl 100U 172-5301

2 units/µl, 250 µl 500U 172-5302

For research purposes only
Store at –20°C

iProof is a high-fidelity DNA polymerase that offers extreme performance for all PCR
applications. Incorporating an exciting new and patented technology, iProof DNA
polymerase brings together a novel Pyrococcus-like enzyme with a processivity enhancing
domain. This allows for the generation of long templates with an accuracy and speed
previously unattainable with a single enzyme. The extreme fidelity of iProof makes it a
superior choice for cloning. The error rate of iProof polymerase is determined to be 4.4 x
10

-7

in iProof HF buffer, which is approximately 50-fold lower than that of Thermus

aquaticus, and 6-fold lower than that of Pyrococcus furiosus.

Storage and Stability

Store iProof™ High-Fidelity DNA Polymerase at -20°C in a constant temperature freezer.
When stored under these conditions, the polymerase is stable for one year after the ship
date.

Kit Contents

Reagent 20U 100U 500U Description

iProof Polymerase 10 µl 50 µl 250 µl iProof™ High Fidelity DNA

Polymerase, 2 units/µl

iProof HF Buffer 1.5 ml 1.5 ml 4 x 1.5 ml 5X HF Buffer, 7.5 mM MgCl

2

iProof GC Buffer 1.5 ml 1.5 ml 4 x 1.5 ml 5X GC Buffer, 7.5 mM MgCl

2

MgCl

2

1.5 ml 1.5 ml 2 x 1.5 ml 50 mM MgCl2solution

DMSO 500 µl 500 µl 500 µl 100% DMSO solution

iProof DNA polymerase is unlike other enzymes. Please read the QuickGuide to
modify your protocol for optimal results.

QuickGuide (See Notes About Cycling Conditions for details)

• Use 98°C for denaturation.

• Anneal at T

m

+3°C (>20nt oligo).

• Use 15–30 sec/kb for extension times. Do not exceed 1 min/kb.

• Use iProof at 0.5–1.0 U per 50 µl reaction. Do not exceed 2 U/50 µl.

• Use 200 µM dNTPs. Do not use dUTP.

• iProof produces blunt end DNA products.

Related Amplification Products From Bio-Rad Laboratories

Reagents for PCR or Real-Time PCR

iProof™ High-Fidelity PCR Kit 172-5331
iProof HF Master Mix 172-5310
iProof GC Master Mix 172-5320
iTaq™ DNA Polymerase 170-8870
iTaq Supermix With ROX 170-8854
iTaq SYBR Green Supermix With ROX 170-8850
iQ™ Supermix 170-8860
iQ SYBR Green Supermix 170-8880
iScript™ cDNA Synthesis Kit 170-8890
iScript Select cDNA Synthesis Kit 170-8896
iScript One-Step RT-PCR Kit with SYBR Green 170-8892
iScript One-Step RT-PCR Kit for Probes 170-8894

For ordering information on larger pack sizes, or to learn more about Bio-Rad amplification
reagents and instruments, visit www.bio-rad.com/amplification/

NOTICE TO PURCHASER: LIMITED LICENSE

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside
the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No.
4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing
patent claims for using only this amount of product for the purchaser’s own internal research. No right under any
other patent claim (such as the patented 5’ Nuclease Process claims in the US Patent Nos. 5,210,015 and
5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind,
including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration,
is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under
Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained
by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404,
USA.

iProof, iTaq, iQ, and iScript are trademarks of Bio-Rad Laboratories..

10002298 Rev B

Bio-Rad Laboratories

2000 Alfred Nobel Drive, Hercules, CA 94547

510-741-1000

5. PCR Additives

The recommended reaction conditions for GC-rich templates include the addition of
3% DMSO which aids in template denaturation. Further optimization of DMSO should
be made in 2% increments. In some cases, DMSO may be used to help relax
supercoiled plasmid DNA. High DMSO concentrations (10%) will require lowering the
annealing temperature by 5.5–6.0°C. Other PCR additives such as formamide,
glycerol, and betaine are also compatible with iProof.

Cycling Conditions

Important Note – Please Read

Due to the novel nature of iProof DNA polymerase, optimal reaction conditions may differ from
standard PCR protocols. iProof works better at elevated denaturation and annealing temperatures
due to higher salt concentration in the reaction buffer.

Typical Thermal Cycling Protocol

Cycle Step Temp. Time Number of Cycles

Initial Denaturation 98°C 30 s 1
Denaturation 98°C 5–10 s
Annealing 45–72°C 10–30 s 25–35
Extension 72°C 15–30 s / kb
Final Extension 72°C 5–10 min 1

Notes About Cycling Conditions

1. Denaturation

Template denaturation should be performed at 98°C. Due to the high thermostability of
iProof, denaturation temperatures greater than 98°C can be used. A 30 s initial
denaturation time is recommended, but this can be extended to 3 min for difficult DNA
templates. Subsequent denaturation should be performed for 5–10 s at 98°C.

2. Annealing

When using iProof, a general rule is to anneal primers (>20 nt) for 10–30 s at +3°C
above the primer with the lowest T

m

. Primer Tmshould be calculated using the nearest­neighbor method as results can vary significantly depending on the method used. For
primers <

20 nt, use an annealing temperature equal to the primer with the lowest T

m

.

3. Extension

Template extension should be performed at 72°C and extension time depends on
amplicon length and complexity. For low complexity DNA (e.g. plasmid, lambda, or
BAC DNA) use 15 s per kb. For high complexity DNA (e.g. genomic DNA) use 30 s per
kb. Do not exceed 1 min per kb for amplicons that are >5 kb.

Component Specifications

Storage buffer

20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% Tween 20, 0.5%
Nonidet P 40, 200 µg/ml BSA and 50% Glycerol

Unit Definition

One unit is defined as the amount of enzyme required to incorporate 10 nmoles of dNTPs into acid­insoluble form at 74°C in 30 minutes under the stated assay conditions.

Enzyme Stability

Each lot of DNA polymerase is tested for stability under normal storage conditions (-20°C). Enzyme
stability is monitored at regular intervals for a two year period after the original assay date.

Reaction Setup

Important Note – Please Read Before Starting

Spin all tubes before opening to improve recovery. Reactions should be set up on ice. Pipet all
components in the order given below. Always add iProof DNA Polymerase last to the reaction as
primer degradation may occur in the absence of dNTPs. It is recommended that you prepare a
master mix for the appropriate number of samples to be amplified.

Volume for Volume for

Component 50 µl reaction 20 µl reaction Final Conc.

5X iProof HF Buffer* 10 µl 4 µl 1X
10 mM dNTP mix 1 µl 0.4 µl 200 µM each
Primer 1** x µl x µl 0.5 µM
Primer 2** x µl x µl 0.5 µM
DNA template x µl x µl
Sterile H

2

0 x µl x µl

iProof DNA Polymerase 0.5 µl 0.2 µl*** 0.02 U/µl
Total Volume 50 µl 20 µl

* For difficult or GC-rich templates, 5X iProof GC Buffer can be used.
** Recommended final primer concentration is 0.5 µM; can range between 0.2–1.0 µM.
*** Enzyme should be diluted to avoid pipeting errors.

Notes About Reaction Components

1. iProof DNA Polymerase

The optimal amount of enzyme depends on the amount of template and the length of
the PCR product. Usually 1 unit of iProof DNA polymerase per 50 µl reaction will give
good results, but optimal amounts could range from 0.5–2 units per 50 µl reaction
depending on amplicon length and difficulty. Do not exceed 2 U/50 µl (0.04 U/µl),

especially for amplicons that are > 5kb.

2. Buffers

Two buffers are provided: 5x iProof HF buffer and 5x iProof GC buffer. The error rate of
iProof polymerase in HF buffer (4.4 x 10

-7

) is lower than that in GC buffer (9.5 x 10-7).
Therefore, the HF buffer should be used as the default buffer for high fidelity
amplification. However, the GC buffer can improve iProof performance on certain
difficult or long templates, i.e. GC rich templates or those with complex secondary
structures. Only use GC buffer when amplification with HF buffer does not provide
satisfactory results.

3. Mg

2+

and dNTP

Mg

2+

concentration is critical since iProof is a Mg2+-dependent enzyme. Excessive

Mg

2+

stabilizes dsDNA, preventing complete denaturation, and can also promote

inaccurate priming. Conversely, insufficient amounts of Mg

2+

can lead to low product

yield. The optimal Mg

2+

concentration also depends on dNTP concentration, the

specific DNA template and the sample buffer composition. The optimal Mg

2+

concentration is 0.5 to 1 mM over the total dNTP concentration for standard PCR. For
optimization, increase or decrease Mg

2+

concentration in 0.2 mM increments.

Only high quality dNTPs should be used. Use of dUTP or other dUTP-derivatives or
analogs is not recommended. Due to the increased processivity of iProof, there is no
advantage to increasing dNTP amounts. For optimal results, use 200 µM dNTPs.

4. DNA Template

General guidelines are 1 pg–10 ng of DNA template in a 50 µl reaction for low
complexity DNA (e.g. plasmid, lambda, or BAC DNA). For high complexity DNA (e.g.
genomic DNA), 50–500 ng of template DNA should be used in a 50 µl reaction.