TI Pfu Plus!

DNA Polymerase
Pfu DNA Polymerase Extremely thermostable proofreading DNA polymerase blend, formulated for efficient
(Pyrococcus furiosus) site-directed mutagenesis and synthesis of ultra wide range of DNA products up to
MODIFIED 20 kb in length.

Cat. No. Size Description:

E1113-01 100 units > tiPfuPlus! DNA polymerase is a new generation “HotStart” enzyme blend. Enzyme
activity is blocked at moderate temperatures and allows room temperature
E1113-02 500 units reaction setup. DNA polymerase actvity is restored during normal PCR cycling
E1113-03 2 500 units conditions.
Storage Conditions: > Use of tiPfuPlus! DNA Polymerase allows for an enormous increase of PCR
Store at -20°C. specificity, sensitivity and yield, as compared to conventional PCR assembly
methods.
> tiPfuPlus! is a modified and optimized hyperthermostable Pfu DNA Polymerase (1)
blended with thermostable polymerisation enhancing factors.
> Ultrapure recombinant enzymes mixture.
> The enzyme catalyzes polymerization of nucleotides into duplex DNA in the 5´ > 3´
direction in the presence of magnesium ions.
> The enzyme exhibits 3´> 5´ proofreading activity, resulting in over 10-fold higher
PCR fidelity than possible with Taq DNA Polymerases (2).
> A constituent of tiPfuPlus! DNA Polymerase, the polymerase-enhancing factor,
enhances PCR product yields and increases target length capability of Pfu DNA
Polymerase.
PCR amplification using EURx ti Pfu Plus! > The enhanced performance of tiPfuPlus! DNA Polymerase allows to use fewer PCR
DNA Polymerase - demonstration of cycles and lower DNA template concentrations, as compared to Pfu DNA
"HotStart" capabilities. Polymerase.
A 0.82 kb amplicon of the human beta-globin > tiPfuPlus! is recommended for use in high-fidelity PCR, PCR of GC-rich sequences
gene was amplified using EURx ti PfuPlus! or problematic secondary structures, primer extension reactions at elevated
DNA Polymerase, 10 x Pfu Buffer and 0.2 mM temperatures, site-directed mutagenesis and cloning of blunt-ended PCR products.
dNTPs in 50 μl reaction volume. To
demonstrate the impact of "Hot Start" on > tiPfuPlus! DNA Polymerase is also recommended for general use in PCR and primer
PCR-yield and PCR-specificity, both, "Hot extension reactions at elevated temperatures to obtain a wide range of DNA
Start" (lanes 1, 2) and non-"Hot Start" products from several hundred bp to over 20 kb.
assays (lanes 3, 4) were incubated at 25°C
for 30 min before performing PCR cycling.
Lane MM: Molecular size marker – Perfect Unit Definition:
100 bp DNA Ladder (Cat. no. E3134).
One unit is defined as the amount of enzyme required to catalyze the incorporation of
Lanes 1,2: PCR amplification reactions using
10 nmoles of dNTP into acid-insoluble material in 30 min at 74°C. The reaction
2.5 U tiPfuPlus! DNA Polymerase(TI-inhibitor
conditions are: 50 mM Tris-HCl (pH 9.0 at 25°C), 50 mM NaCl, 5 mM MgCl2, 200 μM each
mediated “Hot Start”. Reactions were
of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [ 3H]dTTP), 10 μg activated calf
incubated 30 min at 25°C before PCR.
thymus DNA and 0.1 mg/ml BSA in a final volume of 50 μl.
Lanes 3, 4: PCR amplification reactions
using 2.5 U non-"Hot Start" PfuPlus! DNA Storage Buffer:
Polymerase. Reactions were incubated
30 min at 25°C before PCR. 50 mM Tris-HCl (pH 8.2 at 22°C), 0.1 mM EDTA, 1 mM dithiothreitol, 50 % [v/v] glycerol
Lanes 5, 6: PCR amplification reactions and stabilizers.
using 2.5 U non-"Hot Start" PfuPlus! DNA
10 x Reaction Buffer:
Polymerase. Reactions were set up on ice.
10 x Pfu Buffer
The buffer contains 15 mM MgSO4.
Quality Control:
All preparations are assayed for contaminating 3'-exonuclease, and nonspecific single-
and double-stranded DNase activities. Typical preparations are greater than 95 % pure,
as judged by SDS polyacrylamide gel electrophoresis.
References:
1. Lundberg, K., Shoemaker, D., Adams, M., Short, J., Sorge, J. and Mathur E. (1991)
Gene 108, 1.
2. Cline, J., Braham, J. and Hogrefe, H. (1996) Nucleic Acids Res. 24, 3546.
3. Kaledin, A.S., Sliusarenko, A.G. and Gorodetskii, S.I. (1980) Biokhimiya 45, 644.
 TI Pfu Plus! DNA Polymerase
PCR PROTOCOL
Preparation of PCR Reaction: Notes:
Component Volume/reaction Final concentration 1. Concentration Differences: Completely thaw and mix thoroughly all
components of PCR reaction before use to avoid localized differences
10 x Pfu Buffer, contain- 5 μl 1x in salt concentration. This is especially important for magnesium
ing 15 mM MgSO4. solutions, because they form a concentration gradient when frozen.
dNTP mix (5mM each) 2.0-2.5 μl 0.2-0.25 mM each dNTP 2. Room Temperature. Setup reactions at room temperature. Use of
tiPfuPlus! DNA Polymerase allows room temperature reaction setup.
Upstream primer Variable 0.2-0.5 μM Mix well.
Downstream primer Variable 0.2-0.5 μM 3. Cycler Preheating Not Required. Reactions can be placed in a room
temperature thermal cycler.
Template DNA Variable <0.5 μg/50 μl 4. Add Enzyme as Last Component: tiPfuPlus! DNA Polymerase should
be the last component added to the PCR mixture. In the absence of
Sterile double-distilled Variable - dNTPs proofreading (exonuclease) activity of tiPfuPlus! DNA
water Polymerase may degrade primers.
tiPfuPlus! DNA 0.5 μl 2.5 U 5. MgSO4: For tiPfuPlus! DNA Polymerase-based PCR, the standard
Polymerase, 5 U/μl concentration of MgSO4 is 1.5 mM (as provided by the 1 x Pfu Buffer).
In most cases this concentration will produce satisfactory results.
Total volume 50 μl - Should the reaction require inceased Mg 2+ concentrations, use the
supplied 25 mM MgSO4 solution for adjustment.
Adding 1 μl of a 25 mM MgSO4 solution to a total reaction volume of
50 μl will add 25 nmol MgSO4 and thus increase total MgSO4 reaction
concentration in 0.5 mM.
Increasing the MgSO4 concentration enhances PCR yield but
decreases reaction specificity (amplification of more bands, but also
of non-specific bands). Decreasing the MgSO4 concentration
decreases PCR yield but enhances reaction specificity (less bands,
but specific PCR products).
6. dNTP Concentration: The recommended concentration of dNTPs used
in PCR reactions depends on the amplicon length and should be
adjusted empirically. Good results for long targets are usually
achieved by using a dNTP concentration of 0.25 mM (rather than
0.2 mM).
7. Amount of Enzyme: 2.5 U of tiPfuPlus! DNA Polymerase is the
recommended concentration of the enzyme per 50 μl amplification
reaction. For most applications, enzyme will be in excess and will
produce satisfactory results. In some cases it may be necessary to
optimize the enzyme concentration. Excess amounts of enzyme may
Thermal Cycling Conditions for Products up to 6 kb in Size: generate artifacts like as smearing of bands, etc.
Step Temperature Time Number of 8. Additives / PCR Enhancers. In most cases there is no need to add
Cycles additives to the PCR reaction. For some difficult targets such as GC-
Initial 94-95°C 2-5 min 1 rich sequences and targets with complex secondary structures,
Denaturation additives such as DMSO may be included to improve amplification. Use
DMSO in a concentration range between 2-8% [v/v]. The
Denaturation 94-95°C 20-30 s 25-35 recommended starting DMSO concentration (if required) is 3% [v/v].
9. Template DNA Amount: The amount of DNA template required
Annealing 50-68°C 30 s depends on the type of DNA being amplified. Generally, 50-250 ng of
Extension 72°C 1 min/1 kb genomic DNA, 0.1-10 ng of plasmid DNA, 1-20 ng of phage DNA or
10-100 ng of multicopy chromosomal genes is recommended.
Final Extension 72°C 7 min 1
Cooling 4°C Indefinite 1

Thermal Cycling Conditions for Products Larger Than 6 kb in Size: Notes:

Step Temperature Time Number of 1. Annealing: Annealing temperature should be optimized for each
Cycles primer set based on the primer Tm. Optimal annealing temperatures
may be above or below the estimated Tm. As a starting point, use an
Initial 94°C 2 min 1 annealing temperature 5°C below Tm.
Denaturation
2. Long PCR – Primer Requirements: Typical primers for long PCR
Denaturation 94°C 10-15 s 10 amplification reactions have a length of 22-34 bp and should have
annealing temperatures above 60°C to enhance reaction specificity.
Annealing 60-68°C 30 s
3. Long PCR – Short Denaturation Steps: When amplifying long PCR
Extension 68°C 1 min/1 kb products, keep denaturation steps as short as possible and
denaturation temperature as low as possible (try not to exceed 2 min
Denaturation 94°C 10-15 s 25-35 at 94°C during initial denaturation and 10-15 s at 94°C during cycle
denaturation step). Sometimes fragments with high GC-content need
Annealing 60-68°C 30 s higher denaturation temperatures, but keep in mind that the yield
increases when denaturation temperature / duration is decreased.
Extension 68°C 1 min/1 kb
+20 s per 4. Long PCR – Low Elongation Temperature: For PCR products
additional exceeding 6 kb in length use an elongation temperature of 68°C
cycle (rather than 72°C).
5. Long PCR – Extended Elongation Period: For PCR products exceeding
Final Extension 68°C 7 min 1 6 kb in length, an elongation of the extension step (+20 s in each
additional cycle, starting from the 11th cycle) is strongly
Cooling 4°C Indefinite 1 recommended due to loss of processivity of the enzymes blend.
 TI Pfu Plus! DNA Polymerase
SITE DIRECTED MUTAGENESIS PROTOCOL
Preparation of Mutagenesis Reaction: Notes:

Component Volume/reaction Final concentration 1. Concentration Differences: Completely thaw and mix
thoroughly all components of mutagenesis reaction before
10 x Pfu Buffer, contain- 5 μl 1x use to avoid localized differences in salt concentration. It is
ing 1.5 mM MgSO4. especially important for magnesium solutions, because they
form concentration gradients, when frozen.
dNTP mix (5mM each) 2.0-2.5 μl 0.2-0.25 mM of each 2. Room Temperature. Setup reactions at room temperature.
dNTP Use of tiPfuPlus! DNA Polymerase allows room temperature
Mutagenic primer #1 Variable 0.2 μM reaction setup. Mix well.
3. Cycler Preheating Not Required. Reactions can be placed in a
Mutagenic primer #2 Variable 0.2 μM room temperature thermal cycler.
Plasmid DNA Template Variable 5-50 ng 4. Add Enzyme as Last Component: tiPfuPlus! DNA Polymerase
should be the last component added to the mutagenesis
Sterile double-distilled Variable - mixture. In the absence of dNTPs proofreading
water (= exonuclease) activity of tiPfuPlus! DNA Polymerase may
degrade primers.
tiPfuPlus! DNA 0.5 μl 2.5 U
Polymerase, 5 U/μl 5. Amount of Enzyme: 2.5 U of tiPfuPlus! DNA Polymerase is the
recommended concentration of the enzyme per 50 μl
Total volume 50 μl - amplification reaction. For some mutagenesis targets,
further optimization will be required.
6. Target DNA Amount: The mutagenesis protocol usually
requires 5-50 ng of plasmid DNA to achieve satisfactory
results.
7. Placement of Intended Mutation: Both of the mutagenic
primers must contain the intended mutation and anneal to the
same sequence on opposite strands of the plasmid. The
intended mutation should be in the middle of primers,
respectively, with at least 10 bases of correct sequence on
both sides.
8. Amount of Mutagenic Primers: The mutagenic primers should
be used in a concentration of 0.2 μM each per reaction.

Thermal Cycling Conditions: Notes:

Step Temperature Time Number of 1. Annealing Temperature: Adjust the annealing temperature
Cycles accordingly. As a guideline for orientation: Often, the
annealing temperature ranges between 55-60°C, but may
Initial 95°C 1 min 1 differ from these values for certain templates. As a good
Denaturation starting point, use 55°C.
Denaturation 95°C 30 s 18
Annealing X°C 30 s-1 min
Extension 68°C 1 min/1 kb
Final Extension 68°C 7 min 1
Cooling 4°C Indefinite 1