11730017 Pl. QPCR Supermix-UDG

Platinum® Quantitative PCR SuperMix-UDG

Cat. no. 11730-017 Size: 100 reactions

Cat. no. 11730-025 Size: 500 reactions

Store at –20°C

Description

Platinum® Quantitative PCR SuperMix-UDG is a ready-to-use cocktail containing all components, except primers, for the

amplification and detection of DNA in real-time qPCR. The SuperMix combines the automatic “hot-start” technology of

Platinum® Taq DNA polymerase with integrated UDG carryover prevention technology to provide optimal performance with

a variety of qPCR detection technologies, including LUX™ Fluorogenic Primers and TaqMan® probes (1–4). Volumes are

provided for 100 or 500 amplification reactions of 50 μl each.

The SuperMix is supplied at a 2X concentration and contains Platinum® Taq DNA polymerase, Tris-HCl, KCl, 6 mM MgCl2,

400 μM dGTP, 400 μM dATP, 400 μM dCTP, 800 μM dUTP, uracil DNA glycosylase (UDG), and stabilizers.

• Platinum® Taq DNA polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that blocks

polymerase activity at ambient temperatures. Activity is restored after the denaturation step in PCR cycling, providing an

automatic hot start in PCR for increased sensitivity, specificity, and yield (5, 6).

• UDG and dUTP in the SuperMix prevent the reamplification of carryover PCR products between reactions (7). dUTP

ensures that any amplified DNA will contain uracil, while UDG removes uracil residues from single- or double-stranded

DNA, preventing dU-containing DNA from serving as template in future PCRs (8). A UDG incubation step before PCR

cycling destroys any contaminating dU-containing product from previous reactions. UDG is then inactivated by the high

temperatures during normal PCR cycling, thereby allowing the amplification of genuine target sequences.

Magnesium chloride (50 mM) is provided as a separate component to allow adjustment of the magnesium concentration for

optimal performance. ROX Reference Dye is included as a separate component to normalize the fluorescent signal between

reactions, for instruments that are compatible with this option.

Note: This kit is designed for use with fluorogenic primers or probes. For detection using SYBR® Green I dye, we recommend

Platinum® SYBR® Green qPCR SuperMix-UDG (see Additional Products, below).

Component 100-rxn Kit 500-rxn Kit

Platinum® Quantitative PCR SuperMix-UDG 2 × 1.25 ml 12.5 ml

50 mM Magnesium Chloride 1 ml 2 × 1 ml

ROX Reference Dye 100 μl 500 μl

Storage

Components may be stored at either -20ºC or 4ºC. ROX Reference Dye must be stored in the dark.

Quality Control

This product is tested functionally in qPCR using genomic DNA. Kinetic analysis must demonstrate a linear dose response

with decreasing target concentration and detection from 10 pg human genomic DNA. Components are also tested for the

absence of DNase, RNase, and contaminating exonuclease activities.

Additional Products

Product Amount Catalog No.

SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR 50 rxns 1172-050

250 rxns 11752-250

Platinum® Quantitative PCR SuperMix-UDG with ROX 100 rxns 11743-100

500 rxns 11743-500

Platinum® SYBR® Green qPCR SuperMix-UDG 100 rxns 11733-038

500 rxns 11733-046

LUX™ Fluorogenic Primers Visit www.gentaur.com

Part no. 11730.pps Rev. date: 19 Sep 2005

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Guidelines and Parameters

Instrument Compatibility

This kit can be used with a variety of real-time instruments, including but not limited to the ABI PRISM® 7000, 7700, and

7900HT; the ABI 7300 and 7500 Real-Time PCR Systems; the ABI GeneAmp® 5700; the Bio-Rad iCycler™; the Stratagene

Mx3000P®, Mx3005P™, and Mx4000®; the Corbett Research Rotor-Gene™; the MJ Research DNA Engine Opticon™, Opticon® 2,

and Chromo 4™ Real-Time Detector; and the Cepheid Smart Cycler®.

Template

cDNA

For two-step qRT-PCR, use 5 μl of undiluted or 10 μl of diluted cDNA generated from 10 pg to 1 μg of total RNA. For cDNA

synthesis, we recommend SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (see Additional Products, page 1).

Note that detecting high-abundance genes in undiluted cDNA may result in very low CTs in qPCR, leading to reduced

quantification accuracy. Prepare a dilution series of the cDNA template for the most accurate results.

Plasmid and Genomic DNA

Use 100 pg to 1 μg of genomic DNA or 10–107 copies of plasmid DNA in a 10-μl volume. Note that 1 μg of plasmid DNA

contains 9.1 × 1011 copies divided by the plasmid size in kilobases.

Magnesium Concentration

Magnesium chloride is included in the SuperMix at a final concentration of 3 mM. This works well for most targets; however,

the optimal concentration may range from 3 to 6 mM. If necessary, use the 50-mM magnesium chloride provided in the kit to

increase the magnesium concentration, as shown below (the table assumes a 50-μl reaction containing 25 μl of SuperMix):

For a Final MgCl2 Concentration of Add this Volume of 50-mM MgCl2 (per 50-μl Rxn)

4.0 mM 1 μl

5.0 mM 2 μl

6.0 mM 3 μl

Decrease the amount of water in the reaction accordingly.

ROX Reference Dye

ROX Reference Dye can be included in the reaction to normalize the fluorescent reporter signal, for instruments that are

compatible with that option. ROX is supplied at a 25 μM concentration, and is composed of a glycine conjugate of

5-carboxy-X-rhodamine, succinimidyl ester in 20 mM Tris-HCl (pH 8.4), 0.1 mM EDTA, and 0.01% Tween® 20. Use the

following table to determine the amount of ROX to use with a particular instrument (per 50-μl reaction volume):

Instrument ROX per 50-μl reaction Final Concentration

ABI 7000, 7300 7700, 7900HT, and 7900HT Fast 1.0 μl 500 nM

ABI 7500; Stratagene Mx3000™, Mx3005P™, and Mx4000™ 0.1 μl* 50 nM

*To accurately pipet 0.1 μl per reaction, we recommend diluting ROX 1:10 immediately before use and use 1 μl of the dilution.

Note: Platinum® Quantitative PCR SuperMix-UDG with ROX (Catalog nos. 11743-100 and 11743-500) includes ROX in the

SuperMix at a 500 nM final concentration (see Additional Products, page 1)

Multiplexing

In multiplex applications, different reporter dyes are used to label separate primers or probes targeting different genes. For

relative expression studies using multiplex PCR, the amount of primer for the reference gene (e.g., β-actin or GAPDH) should

be limited to avoid competition with the sample gene. In general, the final concentration of the reference gene primer should

be between 25 and 100 nM. A primer titration is recommended for optimal results.

For best results using the following detection systems, the amplicon size should be 80–200 bp.

LUX™ Primers

LUX™ Primers are fluorogenic primers for qPCR that provide high sensitivity, high specificity, multiplexing capability, and

melting curve analysis. LUX™ Primers are available separately and may be

designed for specific targets using the D-LUX™ Designer.

A final concentration of 200 nM per primer is effective for most reactions. Optimal results may require a primer titration

between 100 and 500 nM.

Dual-Labeled Probes

A final probe concentration of 100 nM is effective for most reactions. The optimal concentration may vary between 50 and

500 nM. PCR primers used with probes should be designed according to standard PCR guidelines. A final concentration of

200 nM per primer is effective for most reactions.

Page 3

General Protocol for ABI Instruments

Follow the protocol below for qPCR using either LUX™ Primers or TaqMan® Probes on ABI real-time instruments. Note the

separate cycling conditions for the ABI 7500 in Fast Mode, and the lower amount of ROX Reference Dye required for the ABI

7500 and 7500 Fast systems. This generic protocol may also be used for other real-time instruments.

For protocols for specific instruments: [email protected]. A standard 50-μl reaction size is provided; component

volumes can be scaled as desired (e.g., scaled down to a 20-μl reaction volume for 384-well plates).

1. Program your real-time instrument as shown below. Optimal temperatures and incubation times may vary.

Standard Cycling Program for ABI Instruments

50°C for 2 minutes hold (UDG incubation)

95°C for 2 minutes hold

40 cycles of:

95°C, 15 seconds

60°C, 30 seconds (60 seconds for the 7900HT)

Melting curve analysis (LUX™ Primers only): Refer to

instrument documentation

Fast Cycling Program (for the ABI 7500 in Fast Mode)

Select Fast Mode on the Thermal Profile tab

50°C for 2 minutes hold (UDG incubation)

95°C for 2 minutes hold

40 cycles of:

95°C, 3 seconds

60°C, 30 seconds

Melting curve analysis (LUX™ Primers only): Refer to

instrument documentation

2. Set up reactions as specified below. Volumes for a single 50-μl reaction are listed. For multiple reactions, prepare a

master mix of common components, add the appropriate volume to each tube or plate well, and then add the unique

reaction components (e.g., template). Note: Preparation of a master mix is crucial in qPCR to reduce pipetting errors.

LUX™ Primers Reaction Mix

Component Single rxn

Platinum® Quantitative PCR SuperMix-UDG 25 μl

LUX™ labeled primer, 10 μM 1 μl

Unlabeled primer, 10 μM 1 μl

ROX Reference Dye (optional) 1 μl/0.1 μl*

Template (100 pg to 1 μg of genomic DNA,

10–107 copies of plasmid DNA, or cDNA

generated from 10 pg to 1 μg of total RNA) ≤ 10 μl

DEPC-treated water to 50 μl

TaqMan® Probes Reaction Mix

Component Single rxn

Platinum® Quantitative PCR SuperMix-UDG 25 μl

Forward primer, 10 μM 1 μl

Reverse primer, 10 μM 1 μl

Fluorogenic probe, 10 μM 0.5 μl

ROX Reference Dye (optional) 1 μl/0.1 μl*

Template (100 pg to 1 μg of genomic DNA,

10–107 copies of plasmid DNA, or cDNA

generated from 10 pg to 1 μg of total RNA) ≤ 10 μl

DEPC-treated water to 50 μl

*See the table on page 2 for the amount/concentration of ROX to use for your specific instrument.

3. Cap or seal the reaction tube/PCR plate, and gently mix. Make sure that all components are at the bottom of the

tube/plate; centrifuge briefly if needed.

4. Place reactions in a preheated real-time instrument programmed as described above. Collect data and analyze results.

References

1. Crockett, A.O., and Wittwer, C.T. (2001) Fluorescein-labeled oligonucleotides for real-time PCR: using the inherent quenching of

deoxyguanosine nucleotides. Anal. Biochem. 290, 89–97.

2. Gibson, U. E. M., Heid, C. A., and Williams, P.M. (1996) A novel method for real-time quantitative RT-PCR. Genome Res. 6, 99

3. Nazarenko, I., Lowe, B., Darfler, M., Ikonomi, P., Schuster, D., and Rashtchian, A. (2002) Multiplex quantitative PCR using selfquenched

primers labeled with a single fluorophore. Nucl. Acids Res. 30, e37

4. Nazarenko, I., Pires, R., Lowe, B., Obaidy, M., and Rashtchian, A. (2002) Effect of primary and secondary structure of

oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucl. Acids Res. 30, 2089–2095

5. Chou, Q., Russel, M., Birch, D., Raymond, J., and Bloch, W. (1992) Prevention of pre-PCR mis-priming and primer dimerization

improves low-copy-number amplifications. Nucl. Acids Res. 20, 1717.

6. Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., and Daiss, J.L. (1994) Antibodies as thermolabile switches: high temperature

triggering for the polymerase chain reaction. BioTechnology 12, 506.

7. Longo, M., Berninger, M., and Hartley, J. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain

reactions. Gene 93, 125.

8. Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B., and Sperens, B. (1977) DNA N-glycosidases: properties of uracil-DNA glycosidase

from Escherichia coli. J. Biol. Chem. 252, 3286.

PRISM® and GeneAmp® are registered trademarks of Applera Corporation.

TaqMan® is a registered trademark of Roche Molecular Systems, Inc.

iCycler™, Mx3000P®, Mx3005™, Mx4000®, Rotor-Gene™, DNA Engine Opticon™, Chromo 4™, and Smart Cycler® are trademarks or registered trademarks of their

respective companies.

Page 4

Troubleshooting

Problem Possible Cause Solution

Signals are present in notemplate

controls, and/or

multiple peaks are present in

the melting curve graph

Template or reagents are

contaminated by nucleic

acids (DNA, cDNA)

Use melting curve analysis if possible, and/or run the PCR products on a 4% agarose

gel after the reaction to identify contaminants.

To reduce the risk of contamination, take standard precautions when preparing your

PCR reactions.

Primer dimers or other

primer artifacts are

present

Use melting curve analysis to identify primer dimers by their lower melting

temperature if possible. Use validated primer sets or design primers/probes using

dedicated software programs or primer databases.

Check the purity of your primers by gel electrophoresis. If agarose gels are used, we

recommend cooling the gels before visualization with intercalating dyes.

No amplification curve

appears on the qPCR graph

There is no PCR product Run the PCR product on a gel to determine whether PCR worked. Then proceed to

the troubleshooting steps below.

No PCR product is evident,

either in the qPCR graph or

on a gel

The protocol was not

followed correctly

Verify that all steps have been followed and the correct reagents, dilutions, volumes,

and cycling parameters have been used.

Template contains

inhibitors, nucleases, or

proteases, or has

otherwise been degraded.

Purify or re-purify your template.

Primer design is

suboptimal

Verify your primer selection. Use validated primer sets or design primers/probes

using dedicated software programs or primer databases.

PCR product is evident in

the gel, but not on the qPCR

graph

qPCR instrument settings

are incorrect

Confirm that you are using the correct instrument settings (dye selection, reference

dye, filters, acquisition points, etc.) for your application.

Problems with your

specific qPCR instrument

For instrument-specific tips and troubleshooting: [email protected]

PCR efficiency is above 110% Template contains

inhibitors, nucleases, or

proteases, or has

otherwise been degraded.

Purify or re-purify your template. Inhibitors in the template may result in changes in

PCR efficiency between dilutions

PCR efficiency is below 90% The PCR conditions are

suboptimal

Verify that the amount of primers/probe you are using is correct and that the labeled

primer or probe has not been exposed to direct light.

Verify that the reagents you are using have not been freeze-thawed multiple times

and have not remained at room temperature for too long.

Multiplex reactions:

Primer concentration may

be limiting the rate of the

reaction.

Perform a single-plex reaction using the same primers and template to check

efficiency. Then determine which primer set should be in limiting concentration.

Typically, you should limit the amount of primer for the most abundant gene(s). For

additional multiplex troubleshooting tips: [email protected]

Limited Use Label License No. 1: Thermostable Polymerases

A license under the non-U.S. counterparts of U.S. Patents Nos. 4,683,202, 4,683,195 and 4,965,188 owned by F. Hoffmann-La Roche Ltd, for use in research and development, has an up-front fee component and a running-royalty

component. The purchase price of this product includes a limited, nontransferable license under the running-royalty component to use only this amount of product to practice the Polymerase Chain Reaction (PCR) and related

processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights

to the up-front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process. Rights under the up-front fee component may be purchased from Applied Biosystems or

obtained by purchasing an Authorized Thermal Cycler. This product is also an authorized reagent and may also be used under service sublicenses from Applied Biosystems under the foregoing patents. No right to perform or

offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further

information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Limited Use Label License No. 9: Uracil DNA Glycosylase

The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or

for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or

materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not

for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for

Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its

components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or

its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic,

vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such

product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for

purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-6500.

Limited Use Label License No. 12: SuperMix Products

its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen Corporation and claiming this product based upon the manufacture,

use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its

components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For

information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200. Fax (760) 602-

6500.

Limited Use Label License No. 14: Platinum® products

Licensed to Invitrogen Corporation, under U.S. Patent Nos. 5,338,671; 5,587,287, and foreign equivalents for use in research only.

Limited Use Label License No. 274: 5´ Nuclease Process

The purchase price of this product includes limited, non-transferable rights to use only this amount of the product to practice the 5’ nuclease process solely in the purchaser’s own research and development activities under the

following patents of Roche Molecular Systems, Inc. or F. Hoffmann-La Roche Ltd, (“Roche”): US. Patents Nos. 5,210,015, 5,487,972, 5,476,774, and 5,219,727, and corresponding patent claims outside the United States. This

product is also an Authorized Core Kit for use with service sublicenses from Applied Biosystems under the foregoing patents. No right to perform the 5’ nuclease process for any other purpose or to offer commercial services of

any kind using the 5’ nuclease process, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is hereby granted expressly, by implication, or by estoppel. No rights

are granted under US. Patent No. 5,804,375 or counterpart claims outside the United States, covering 5’ nuclease reaction mixtures, or under any patent claim covering probes useful in the 5’ nuclease process, expressly, by

implication or by estoppel. Rights under U.S. Patent No. 5,804,375 and counterpart claims outside the United States and under probe patent claims may be purchased from Applied Biosystems, or may be obtained by purchasing

probes (from Applied Biosystems or other licensees) conveying those rights. Further information on purchasing licenses to practice these patented processes may be obtained by contacting the Director of Licensing, Applied

Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.