What is the difference between the TaqMan® Universal PCR Master Mix Cat. No. 4304437 and Cat. No. 4324018?

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Both part numbers for TaqMan® Universal PCR Master Mix are supplied at a 2X concentration and contain the following components: AmpliTaq Gold® DNA polymerase, dNTPs with dUTP, Passive Reference (ROX), and optimized buffer components. However, the part number 4304437 also contains AmpErase® UNG and is not suitable to be used for One Step RT PCR.

Answer Id: 1581

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If I inadvertently put the TaqMan® Universal Master Mix (Cat. No. 4304437) in the freezer at -20 degrees C, can I still use it for my experiments?

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Although it is not recommend to freeze the Universal Master Mix, testing has shown that if it is frozen and then thawed, you should not experience any difficulties. If the Universal Master Mix is frozen, make sure that it is thoroughly thawed and mixed prior to use, followed by proper storage at 4 degrees C.

Answer Id: 1550

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What Applied Biosystems® reagents and instruments can I use with TaqMan® Gene Expression Assays?

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The TaqMan® Gene Expression Assays are optimized to work with one of the TaqMan® qPCR Master Mixes that include TaqMan® Universal PCR Master Mix, No AmpErase® UNG(P/N 4324018), TaqMan® Universal PCR Master Mix with AmpErase UNG (P/N 4304437), TaqMan® Universal PCR Master Mix II, No AmpErase® UNG (P/N 4440040), TaqMan® Universal PCR Master Mix II, with AmpErase® UNG (P/N 4440038) or TaqMan® Gene Expression Master Mix (P/N 4369016) and with your cDNA. It is recommended to use the High Capacity cDNA Archive Kit (P/N 4322171) for converting your RNA to cDNA. TaqMan® Gene Expression Assays are capable of being run on all Applied Biosystems® Real-Time PCR Systems models including 7000, 7300, 7500, 7500 Fast, 7700, 7900HT, StepOne™, StepOnePlus™ and ViiA™ 7 Instruments.

Answer Id: 1834

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Why does amplification efficiency decrease or plateau in the late cycles?

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Three limitations may contribute to the decrease in amplification efficiency:

1. Plateau occurs in PCR because of the progressive reduction in the efficiency of the primer-template complex formation due to product reannealing. The primers in PCR are always present at high excess and the kinetics of reannealing are determined by the molecule in the greatest concentration. Typically, total initial PCR primer concentrations are 2x10E-6 M. However, as the reaction proceeds, the DNA target concentration increases and the primer concentration decreases, albeit only slightly, to 1.4x10E-7 M to 2x10E-6 M, and it takes a longer time for the primer-template complex to form. If the product strands have a chance to reanneal to themselves, forming a very stable complex, the primers will not have a binding site for extension and doubling may not occur. This snap-back of product strands reannealing to themselves can occur in a few seconds (or less) at high DNA concentrations.

2. Primer concentration depletion. One cause of primer depletion is the formation of primer artifacts and other spurious, non-specific amplification products.

3. Insufficient enzyme concentration or polymerization time in late cycles. In a typical PCR amplification, 2.0 - 2.5 U or roughly 8x10E10 molecules of AmpliTaq® DNA Polymerase are used. In plateau there are roughly 1x10E12 template copies leaving less than one molecule of AmpliTaq® DNA Polymerase per primer-template complex. One way to overcome the limiting polymerase is to increase the extension time in the later cycles of the PCR amplification.

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Can dU-containing DNA produced by amplification with dUTP be cut by restriction enzymes?

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You will need to confirm with the manufacturer of the restriction enzyme you are using to find out whether or not it can recognize dUTP-containing DNA.

Answer Id: 1411

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What is the magnesium chloride concentration in the TaqMan® PCR Universal Master Mix? Why is it not listed in the product documentation?

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The concentrations of the components in the TaqMan® Universal Master Mix are a key part of the product's high-performance benefits, and therefore are considered proprietary. That is why the component concentrations are not included in the product description. The master mix was designed to be used with our TaqMan assays and the universal cycling conditions that they all function at, which eliminates the need for optimization. However, as a good reference point the optimal Mg concentration for the Amplitaq Gold, (which is the Taq polymerase in the Universal Master Mix), is 1.5 mM - 4 mM.

Answer Id: 1582

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b710915795b9e9c02cf10d6d2bdb688c_FAQ

Can I use TaqMan® 2X Universal PCR Master Mix (including AmpErase® UNG with TaqMan® MicroRNA Assays?

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TaqMan® MicroRNA Assays protocol (Cat. No. 4364031) recommends TaqMan® 2X Universal PCR Master Mix, No AmpErase® UNG (Cat. No. 4324018). However, all TaqMan® MicroRNA Assays can be used with TaqMan® 2X Universal PCR Master Mix, with AmpErase® UNG (Cat. No. 4304437).

Answer Id: 2770

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Can I use the TaqMan® Universal PCR Master Mix with Amperase® UNG when running a TaqMan® Drug Metabolism Genotyping Assay?

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Yes, you can use either the TaqMan® Universal Master Mix with Amperase UNG (P/N 4304437) or the TaqMan® Universal Master Mix without Amperase UNG (P/N 4324018). When using the TaqMan® Universal Master Mix with Amperase UNG, you should add a 50C x 2 min holding stage prior to 95C x 10 min holding stage. When using the TaqMan® Universal Master Mix without Amperase UNG, delete the initial 2 minute 50 C hold stage.

Answer Id: 2429

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a9365bd906e11324065c35be476beb0c_FAQ

Can I check the end product of a TaqMan® Gene Expression Assay by running an agarose gel?

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Yes, you can check the end product on an agarose gel. If you use the TaqMan® Universal PCR Master Mix (including AmpErase® UNG, Cat. No. 4304437), we recommend running the gel immediately after the PCR, since the trace amount of AmpErase® UNG may digest the end product that contains double-stranded DNA with dUTP incorporated. The average length of amplicon ranges from 50-150 bp. The exact amplicon length is indicated on the detailed page of each assay on our website. You can get to that page by clicking on the Assay ID; the amplicon information is listed in the section "Assay Details".

Answer Id: 2464

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What are the components of a TaqMan® Gene Expression Assay?

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Each TaqMan® Gene Expression Assay consists of two unlabeled PCR primers and a FAM™ dye-labeled TaqMan® MGB probe (non-fluorescent quencher). All the components are QC tested and combined into a 20X formulation. Purchase of a TaqMan Gene Expression Assay does not include the TaqMan® Universal PCR Master Mix (P/N 4304437).

Answer Id: 1833

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What is the purpose of the RNase P Detection Reagents Kit (Cat. No. 4316831), and what is provided in the kit?

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RNase P is a single-copy gene encoding the RNA moiety for the RNase P enzyme, and it is an excellent gene to use as a reference/control for Copy Number Variation (CNV) assays. The TaqMan® RNase P Detection Reagents Kit contains a 20X mix of primers and probe (FAM™ dye-labeled) that will detect and quantitate genomic copies of the human RNase P gene. The primers and probe are designed according to TaqMan® chemistry guidelines for quantitation, and utilize the universal thermal cycling parameters. This kit is used to perform a 5' nuclease assay with TaqMan® Universal PCR Master Mix (Cat. No. 4304437) or the TaqMan® Core PCR Reagents Kit (Cat. No. N808-0228) with genomic, plasmid or complementary DNA (cDNA) templates.

The complete components of the TaqMan® RNase P Detection Reagents Kit, Cat. No. 4316831, are: 20X RNase P Primer-Probe (FAM™/TAMRA™ probe) Mix, 1 tube Human Genomic Control DNA, 10 ng/ul (100 ul), and the Product Insert with an overview and procedure for the use of the kit.

Answer Id: 1421

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