PfuTurbo DNA Polymera AD
Catalog #600255, 600257, and 600259
600255-12, Revision A.01
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让我欢喜让我忧M ATERIALS P ROVIDED
Quantity
Materials provided Catalog #600255 Catalog #600257 Catalog #600259
PfuTurbo DNA Polymera 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 polymera AD* features a reformulated buffer system for incread economy with the
same high performance as the original Stratagene PfuTurbo DNA polymera for robust, high-fidelity PCR. As with the original PfuTurbo DNA polymera, PfuTurbo DNA polymera AD is a mixture of cloned Pfu DNA polymera and the exclusive thermostable ArchaeMaxx polymera-enhancing factor1 that enhances PCR product yields and increas target length capability without altering DNA replication fidelity. PfuTurbo DNA polymera AD can be ud to amplify complex genomic DNA targets up to 19 kb and vector targets up to 15 kb in length. PfuTurbo DNA polymera AD exhibits the same low error rate as Pfu DNA polymera of 1–3 errors per 106 bas, while Taq DNA polymera exhibits an error rate of 8–9 errors per 106 bas 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 polymera 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
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)
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 ction 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 Polymera 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 polymera 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 DNa-, RNa-, and pr
otea-free mineral oil (Sigma, St.
Louis, Missouri).
PfuTurbo DNA Polymera AD 600255-12 Revision A.01
© Agilent Technologies, Inc. 2009.
3. Perform PCR using optimized cycling conditions. Suggested cycling parameters for PfuTurbo DNA polymera AD-bad 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) agaro gel.
PCR Cycling Program for PfuTurbo DNA Polymera 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
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1
95°C a
1 minute 1 minute 95°C
30 conds 1 minute Primer T m – 5°C 30 conds 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疾风知劲草的意思
1
1
92°C b 2 minutes 1 minute 92°C b
10 conds 1 minute Primer T m – 5°C 30 conds 1 minute 2 10
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2 minutes per kb 2 minutes per kb 92°C
10 conds 1 minute Primer T m – 5°C 30 conds
1 minute
3 20 68°C
2 minutes per kb plus 10 conds per cycle
2 minutes per kb plus 10 conds per cycle
a
Denaturing temperatures above 95°C are recommended only for GC-rich templates. b
For cDNA targets, u 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 polymera AD. Optimize the PCR program denaturation time (typically 30–60 conds 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. U 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.夏天有什么花开
• 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 increa performance for some targets/cycling programs.
• PCR cloning: If generating PCR fragments for cloning applications, u the StrataClone Blunt PCR Cloning Kit (Catalog #240207) or another blunt PCR cloning strategy. PfuTurbo DNA polymera AD does not exhibit terminal deoxynucleotidyltransfera (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 purpo, are provided by Agilent. Agilent shall have no liability for any direct, indirect, conquential, or incidental damages arising out of the u, the results of u, or the inability to u 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. Purcha of this product conveys to the purchar only the non-transferable right under the
patents to u the product for rearch u only by the purchar . No rights are granted to the purchar hereunder to ll, modify for resale or otherwi transfer this product, either alone or as a component of another product, to any third party. Agilent rerves all other rights, and this product may not be ud in any manner other than as provided herein. For information on obtaining a licen to u this product for purpos other than rearch, plea 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., Hann, 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.
创意空间
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
/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.
