Bio-Rad iProof™ High-Fidelity DNA Polymerase - Manual (Page 2)

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 T
m
should be calculated using the nearest-
neighbor 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 acid-
insoluble 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 Mg 2+ -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.