PfuTurbo DNA Polymerase AD
Catalog #600255, 600257, and 600259
600255-12, Revision A.01
M ATERIALS P ROVIDED
Quantity
Materials provided Catalog #600255 Catalog #600257 Catalog #600259
PfuTurbo DNA Polymerase AD 100 U 500 U 1000 U
10× Cloned Pfu Reaction Buffer AD    1 ml    2 × 1 ml    4 × 1 ml
Storage: Store at –20°C upon receipt.
I NTRODUCTION
PfuTurbo DNA polymerase AD* features a reformulated buffer system for increased economy with the
same high performance as the original Stratagene PfuTurbo DNA polymerase for robust, high-fidelity PCR. As with the original PfuTurbo DNA polymerase, PfuTurbo DNA polymerase AD is a mixture of cloned Pfu DNA polymerase and the exclusive thermostable ArchaeMaxx polymerase-enhancing factor1 that enhances PCR product yields and increases target length capability without altering DNA replication fidelity. PfuTurbo DNA polymerase AD can be used to amplify complex genomic DNA targets up to 19 kb and vector targets up to 15 kb in length. PfuTurbo DNA polymerase AD exhibits the same low error rate as Pfu DNA polymerase of 1–3 errors per 106 bases, while Taq DNA polymerase exhibits an error rate of 8–9 errors per 106 bases in the same assay.2
O PTIMIZATION P ARAMETERS (50µL REACTION VOLUME)
Parameter Genomic DNA Targets ≤6 kb
or Vector DNA Targets ≤10 kb
Genomic DNA Targets >6 kb
or Vector DNA Targets >10 kb  cDNA Targets
Extension time    1 minute per kb    2 minutes per kb    2 minutes per kb PfuTurbo
DNA polymerase AD    2.5 U    5.0 U    5.0 U
Input template 50–100 ng genomic DNA;
100 pg–30 ng vector DNA 200–250 ng genomic DNA;
100 pg–30 ng vector DNA
1–2 μl cDNA (from cDNA
synthesis reaction)
Primers (each) 100–200 ng (0.2–0.5 μM) 200 ng (0.5 μM) 100–200 ng (0.2–0.5 μM) dNTP concentration 100–250 μM each dNTP 500 μM each dNTP 100–250 μM each dNTP
Final reaction buffer conc.    1.0×    1.5× for genomic DNA
or 1.0× for vector DNA 1.0× (supplemented with  1 mM MgSO
4
)
Denaturing temperature 95°C 92°C 95°C
Extension temperature 72°C 68°C 68°C
PCR P ROTOCOL
The reaction conditions given here are for amplification of a typical single-copy chromosomal target of ≤6 kb. See the Optimization Parameters section for guidelines on amplifying longer targets, vector DNA targets, or cDNA targets. The reaction conditions are for one reaction and must be adjusted for multiple samples. The final volume of each reaction is 50 µl.
1. Add the components in order into sterile thin-walled PCR tubes while mixing gently.
Reaction Mixture for a Typical Single-Copy Chromosomal Locus PCR Amplification (≤6 kb)
Component Amount per reaction
Distilled water (dH
2
O) 40.6 μl
10× Cloned Pfu Reaction Buffer AD a    5.0 μl
dNTP mix (25 mM each dNTP)  0.4 μl
DNA template (100 ng/μl)      1.0 μl
Primer #1 (100 ng/μl)      1.0μl b
Primer #2 (100 ng/μl)      1.0μl b
PfuTurbo DNA Polymerase AD (2.5 U/μl)      1.0μl c
Total reaction volume    50 μl
a The 10× buffer provides a final 1× Mg2+ concentration of 2 mM. When amplifying cDNA, add Mg2+ to a final 1× concentration of 3 mM.
(For example, Mg2+ concentration may be adjusted to 3 mM in the final 50-μl reaction volume by adding 2 μl of a PCR-grade
25 mM MgSO
4 solution and reducing the amount of dH
2
O to 38.6 μl.)
b The recommended primer concentration of 0.2–0.5 μM corresponds to 100–200 ng for a typical 18- to 25-mer oligonucleotide primer in a
50-μl reaction volume.
c The amount of PfuTurbo DNA polymerase AD varies depending on the length of the PCR target. The standar
d amount for vector targets up to
10 kb and genomic targets up to 6 kb in length is 1 μl (2.5 U).
2. If the extension time is >30 minutes, overlay each reaction with ~50 µl of DNase-, RNase-, and pro
tease-free mineral oil (Sigma, St.
Louis, Missouri).
PfuTurbo  DNA Polymerase AD 600255-12    Revision A.01
© Agilent Technologies, Inc. 2009.
3. Perform PCR using optimized cycling conditions. Suggested cycling parameters for  PfuTurbo  DNA polymerase AD-based PCR are
provided below. (Optimized cycling parameters are not necessarily transferable between thermal cyclers. Consult the instrument manufacturer’s recommendations if further optimization of cycling parameters is required.) Analyze the PCR amplification products on a 0.7–1.0% (w/v) agarose gel.
PCR Cycling Program for PfuTurbo  DNA Polymerase AD
Genomic DNA Targets ≤6 kb and Vector DNA Targets ≤10 kb Segment
Number of cycles Temperature Duration–Single Block Thermocycler
Duration–Stratagene  RoboCycler Thermocycler
1
pines1
95°C a
1 minute      1 minute 95°C
30 seconds      1 minute Primer T m  – 5°C 30 seconds      1 minute 2 30  72°C
1 minute per kb      1 minute per kb 3      1 72°C
10 minutes
10 minutes
Genomic DNA Targets >6 kb, Vector DNA Targets >10 kb, and cDNA Targets Segment
Number of cycles
Temperature
Duration–Single Block Thermocycler
Duration–Stratagene  RoboCycler Thermocycler
1
1
92°C b      2 minutes      1 minute 92°C b
10 seconds      1 minute Primer T m  – 5°C 30 seconds      1 minute 2 10
68°C
2 minutes per kb      2 minutes per kb 92°C
10 seconds      1 minute Primer T m  – 5°C 30 seconds
1 minute
3 20 68°C
2 minutes per kb plus 10 seconds per cycle
2 minutes per kb plus 10 seconds per cycle
a
Denaturing temperatures above 95°C are recommended only for GC-rich templates. b
For cDNA targets, use a denaturation temperature of 95°C.
T ROUBLESHOOTING AND A PPLICATION N OTES
• Low yield: If PCR product yields are lower than expected, optimize the extension time, amount of DNA template (excess DNA can inhibit PCR), and amount of PfuTurbo  DNA polymerase AD. Optimize the PCR program denaturation time (typically 30–60 seconds at 95°C is sufficient; prolonged denaturation steps may damage the template DNA) and the annealing temperature. Extraneous salts contributed by primer or template DNA solutions may inhibit the PCR reaction. Use high-quality, gel purified primers and highly-purified template DNA.
• Multiple bands: The annealing temperature may require optimization. Typically annealing temperatures will range between 55°C and 72°C. Try adding Stratagene Perfect Match PCR Enhancer to improve specificity. Redesign primers.
• PCR adjuncts and cosolvents : Including a cosolvent, such as 1–10% (v/v) DMSO or 5–20% glycerol, or an adjunct, such as 1–5% formamide or 0.01–1 U of Stratagene Perfect Match PCR Enhancer, in the PCR reaction may increase performance for some targets/cycling programs.
• PCR cloning: If generating PCR fragments for cloning applications, use the StrataClone Blunt PCR Cloning Kit (Catalog #240207) or another blunt PCR cloning strategy. PfuTurbo  DNA polymerase AD does not  exhibit terminal deoxynucleotidyltransferase (TdT) activity, which is characterized by the addition of nontemplate-directed nucleotide(s) at the 3´ end of PCR-generated fragments.
L IMITED P RODUCT W ARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use t
his product.
L IMITED L ABEL L ICENSE FOR P FU -C ONTAINING DNA  P OLYMERASE P RODUCTS
This product is covered by the claims of one or more of the following U.S. Patents: 5,545,552; 5,556,772; 5,866,395; 5,948,663; 6,183,997; 6,444,428, 6,489,150; 6,734,293; 7,045,328, and patents pending. Purchase of this product conveys to the purchaser only the non-transferable right under these
patents to use the product for research use only by the purchaser . No rights are granted to the purchaser hereunder to sell, modify for resale or otherwise transfer this product, either alone or as a component of another product, to any third party. Agilent reserves all other rights, and this product may not be used in any manner other than as provided herein. For information on obtaining a license to use this product for purposes other than research, please contact Stratagene Products Division, Business Development, 11011 North Torrey Pines Road, La Jolla, California 92037. Phone (858) 535-5400.
R EFERENCES
1. Hogrefe, H. H., Hansen, C. J., Scott, B. R. and Nielson, K. B. (2002)  Proc Natl Acad Sci U S A  99(2):596–601.
2. Cline, J., Braman, J. C. and Hogrefe, H. H. (1996)  Nucleic Acids Res  24(18):3546-51.
E NDNOTES
* U.S. Patent Nos. 7,045,328,  6,734,293,  6,489,150,  6,444,428,  6,183,997,  5,948,663,  5,866,395,  5,545,552 and patents pending.
MSDS  I NFORMATION
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
www.stratagene/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s in a print-ready format. MSDS documents are not included with product shipments.

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