Thermo Scientific PCR Reagent Selection Guidefscimage.fishersci.com/cmsassets/downloads/segment/... · suit any workflow. This complete portfolio of PCR solutions is the result of

Thermo Scientific PCR Reagent Selection Guide

042 www.thermo.com/pcrsimplified 3042 www.thermo.com/pcrsimplified 3

Your work is challenging. Choosing the right PCR reagent shouldn’t be.

The Thermo Scientific PCR reagent line is a concise portfolio of powerful enzymes built to satisfy the broad range of demanding PCR applications. Whether you are performing routine detection or site-directed mutagenesis, our line provides a clear solution for success with your application. To deliver the ultimate in assay flexibility, our enzymes are supported by a range of convenient formats and custom services to suit any workflow.

This complete portfolio of PCR solutions is the result of over 20 years of ongoing development activities, which include both in-house research and collaborations with industrial and academic partners. Our commitment to continual innovation enables us to provide you with the best PCR systems, now and in the future.

Look to the Thermo Scientific reagent line for a clear solution to your PCR challenge.

PCR Simplified

Standard

High Yield

Hot Start

High Fidelity

Long Range

RT-PCR

044 www.thermo.com/pcrsimplified 5

Legend

• Recommended Enzyme

• Suitable Alternative

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18 PCR Plastics & Custom Services

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High Fidelity PCR

PCR applications requiring highly accurate amplification,

to produce accurate PCR products with minimal

sequence errors.

Long Range PCR

PCR applications requiring robust, accurate amplification of target sequences greater

than 5 kb in length.

RT-PCR

PCR applications that target RNA templates and require

reverse transcription prior to PCR amplification.

High Yield PCR

PCR applications requiring increased yield and sensitivity,

to produce large amounts of PCR product.

Standard PCR

Routine PCR applications requiring consistent and reliable

Taq performance. Yes/No sequence detection.

Taq DNA PolymeraseThermoPrime

Taq DNA PolymeraseThermo-Start

Taq DNA PolymeraseVerbatim High Fidelity

DNA PolymeraseExtensor Long Range

Enzyme Blend

ApplicationsRoutine PCR • • • •Sequencing • • • •Multiplexing •Genotyping • • • •

Library Construction • • • •Cloning • • • •1 •

Mutagenesis •Long PCR • •

Pathogen Detection • • • •Single-cell PCR • • •

SpecificationsGC Rich Templates (w/ High Perf. Buffer) (w/ GC Buffer) (w/ Buffer 2)

Low Copy Templates

Long Templates

Amplicon Size 5 kb 5 kb 5 kb 10 kb 20 kb (w/ Buffer 2)

Proofreading Activity

Blunt or 3’-A Ends 3’-A 3’-A 3’-A Blunt1 3’-A

PerformanceYield

Specificity

Sensitivity

Accuracy / Fidelity


Hot-Start PCR

Challenging PCR applications requiring maximum specificity

and sensitivity, to detect rare or complex targets.

Thermo Scientific PCR Enzyme Portfolio

1 Enzyme creates blunt ends, A overhangs must be generated prior to ligation into T/A vector

2-Step RT-PCRUsed to create a complete cDNA pool. Aliquots from this pool can be used in separate PCR assays to study multiple genes.

Verso cDNA Synthesis Kit can be paired with any PCR Kit for complete 2-Step RT-PCR.

1-Step RT-PCRReverse transcription and amplification steps are carried out in a single reaction tube. Convenient, fast analysis for studying single gene targets.

Verso 1-Step RT-PCR Kits are available in two formats: High Yield (w/ ThermoPrime) and Hot-Start (w/ Thermo-Start)

Verso Reverse Transcriptase

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Application Guide:Achieve the best results by choosing an enzyme system designed for success with your application.


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Custom

Aliquoting Services

To meet the demands of high-throughput applications, our PCR

reagents can be supplied pre-aliquoted into ABgene PCR plastics, eliminating PCR

set-up steps and allowing hundreds of reactions to be prepared in minutes.


Standard PCRTaq DNA PolymerasePCR is a highly sensitive reaction, and impurities in the enzyme mix can cause reduced efficiency or complete reaction inhibition. Any PCR application, no matter how routine, requires a Taq polymerase that is highly purified, and performs consistently from batch to batch.

Thermo Scientific Taq DNA Polymerase is a 94 kDA ultra-pure recombinant thermo-stable DNA polymerase obtained by high level expression of the Taq DNA polymerase gene in Escherichia coli. Thermo Scientific Taq DNA Polymerase is licensed and optimized for PCR and is able to amplify DNA fragments up to 5 kb. Stringent quality controls make this a highly reliable Taq DNA polymerase for use in all routine PCR and RT-PCR applications.

Benefits:

• Reliable performance in all routine PCR applications

• Extensive QC testing ensures batch-to-batch consistency

52.2800653295Genomic DNA Target length >>

76

kb bp

Figure 1: Thermo Scientific Taq delivers successful amplification over a range of target lengths

PCR amplification of 6 target lengths of human genomic DNA (76 bp, 295 bp, 653 bp, 800 bp, 2.2 kb, and 5 kb)each using 12.5 ng of genomic DNA template.

ENZYME KIT

DNA Polymerase / 10X PCR Buffer / MgCl2

COMPLETE PCR KIT

DNA Polymerase / 10X PCR Buffer / MgCl2 / 20 mM dNTPs

PCR Buffer containing DNA Polymerase, MgCl2 and dNTPs

ENZYME KIT WITH REDDYMIX BUFFER

DNA Polymerase /10X ReddyMix Buffer / MgCl2

COMPLETE PCR KIT WITH REDDYMIX BUFFER

DNA Polymerase/ 10X ReddyMix Buffer / MgCl2 / 20 mM dNTPs

Enzyme Kits are the most basic format. Kits include the enzyme, PCR buffer, and 25 mM MgCl2 solution, each in separate vials. dNTPs must be purchased separately.

Complete PCR Kits include all the components needed to perform PCR including an HPLC-purified dNTP mix. Complete PCR kits are a convenient option that still allows for MgCl2 optimization.

Master Mix formats consist of a single master buffer containing all the components needed for PCR. Master Mixes are the most convenient format and significantly reduce assay set-up steps.

Format Options

Save time and effort with ReddyMix Versions!

ReddyMix buffers contain an inert red dye that eases PCR set-up, and a gel loading precipitant that allows for direct loading onto agarose gels. The red dye aids in error free pipetting, and the precipitant allows the completed PCR reaction to be directly loaded onto agarose gels eliminating the need for separate gel loading buffer.

ReddyMix formats ease reaction set-up and reduce handling time leading to faster results and improved reproducibility. For the ultimate in convenience and simplified assay set-up, choose ReddyMix Master Mix (see figure below).

Thermo Scientific PCR Enzyme Systems are available in a range of formats and pack sizes to suit any workflow.

Choose Your Preferred Format:

Cat. No. Units Reactions

AB-0192/A 250 U 400 x 25 μl

AB-0192/B 2500 U 4000 x 25 μl

w/ MgCl2 Pre-mixed into PCR Buffer

AB-1192/A 250 U 400 x 25 μl

AB-1192/B 2500 U 4000 x 25 μl

Cat. No. Units Reactions

AB-0192/A/N 250 U 400 x 25 μl

AB-0192/B/N 2500 U 4000 x 25 μl


AB-1192/A/N 250 U 400 x 25 μl

AB-1192/B/N 2500 U 4000 x 25 μl


ENZYME KIT

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COMPLETE PCR KIT


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MgCl2 Concentration:All buffers premixed with MgCl2 will be at 1.5 mM concentration in the final reaction.

Unit Definition:One unit of enzyme is defined as the amount that will incorporate 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 74°C under the following analysis conditions:25 mM TAPS, pH 9.3 (at 25°C)50 mM KCl2 mM MgCl21 mM β-mercaptoethanol250 μM of each: dCTP, dGTP, dTTP250 μM [3H] dATP (0.05 Ci/mmol)1.25 μg/μl activated salmon sperm DNA

Detailed Product InformationFor more information or to download the product manual, please visit:www.thermo.com/taq

REDDYMIX MASTER MIXREDDYMIX MASTER MIX

ReddyMix Master Mix containing DNA Polymerase, MgCl2 and dNTPs

STANDARD PCR KIT

Calculate and mix components to prepare master buffer

Load Gel &Analyze

REDDYMIX MASTER MIX

Dispense MasterMix, Templateand Primers

Run PCRProtocol

Load Gel &Analyze

Standard PCR Kit VS ReddyMix Master Mix

Choose ReddyMix Master Mix format for the most streamlined PCR workflow

Thermo Scientific PCR Reagents

Dispense MasterMix, Templateand Primers

Run PCRProtocol

Add gel loading buffer to each sample


Available

Pre-Aliquoted into ABgene

PCR Plastics

Notes

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High Yield PCRThermoPrime Taq DNA PolymerasePCR assays using standard Taq DNA polymerase and basic buffer formulations are subject to a loss in polymerase activity over the multiple high temperature steps of PCR cycling. This incremental loss of activity can result in decreased end yield and reduced assay sensitivity.

ThermoPrime Taq DNA Polymerase exhibits enhanced thermal stability giving extended polymerase activity over standard Taq DNA polymerase formulations. A proprietary buffer component protects ThermoPrime during high temperature steps, ensuring optimal performance throughout the PCR reaction, leading to increased end yields. Sustained enzyme activity also helps maintain reaction efficiency and exponential amplification, making ThermoPrime an ideal enzyme for comparative analysis of gene expression. 800 bp >>

Genomic DNA Template (ng) >> 100 50 25 12.5 6.3 3.2 1.6 0.8

Benefits:

• Enhanced enzyme stability for increased yield

• Advanced buffering system minimizes PCR optimization steps

• Convenient Master Mix formats

Cat. No. Pack Size

AB-0301/A/N 400 x 25 μl rxns

AB-0301/B/N 4000 x 25 μl rxns


AB-1301/A/N 400 x 25 μl rxns

AB-1301/B/N 4000 x 25 μl rxns

Red Hot Taq Version (Red Dye w/Enzyme)

AB-0406/A/N 400 x 25 μl rxns

AB-0406/B/N 4000 x 25 μl rxns


AB-1406/A/N 400 x 25 μl rxns

AB-1406/B/N 4000 x 25 μl rxns

Cat. No. Pack Size

1.1X Concentration

AB-0575/A 160 x 25 µl rxns

AB-0575/B 1600 x 25 µl rxns

2X Concentration

AB-0575/DC/A 160 x 25 µl rxns

AB-0575/DC/B 1600 x 25 µl rxns

Cat. No. Pack Size

AB-0790/A 400 x 25 μl

AB-0790/B 4000 x 25 μl


AB-0785/A 400 x 25 μl

AB-0785/B 4000 x 25 μl

Cat. No. Pack Size

AB-0790/A/N 400 x 25 μl rxns

AB-0790/B/N 4000 x 25 μl rxns


AB-0785/A/N 400 x 25 μl rxns

AB-0785/B/N 4000 x 25 μl rxns

Cat. No. Pack Size

1.1X Concentration

AB-0575/LD/A 160 x 25 µl rxns

AB-0575/LD/B 1600 x 25 µl rxns

2X Concentration

AB-0575/DC/LD/A 160 x 25 µl rxns

AB-0575/DC/LD/B 1600 x 25 µl rxns

Red Dye in enzyme mix helps helps verify enzyme addition during reaction set-up


Figure 3: Easily detect pipetting errors with Red Hot Taq formats and ReddyMix kits and master mixes.

Red Hot formats include an inert red indicator dye in the enzyme mix, and ReddyMix kits contain an inert red dye in the buffer (in addition to a gel loading precipitant).

Figure 2: ThermoPrime Taq DNA Polymerase delivers high yield over a range of template concentrations.

PCR amplification of 8 concentrations of human genomic DNA (100, 50, 25, 12.5, 6.3, 3.2, 1.6 and 0.8 ng) using a 800 bp β-actin gene fragment as the target.

NotesCat. No. Pack Size

AB-0301/A 400 x 25 μl rxns

AB-0301/B 4000 x 25 μl rxns


AB-1301/A 400 x 25 μl rxns

AB-1301/B 4000 x 25 μl rxns

Red Hot Taq Version (Red Dye w/Enzyme)

AB-0406/A 400 x 25 μl rxns

AB-0406/B 4000 x 25 μl rxns


AB-1406/A 400 x 25 μl rxns

AB-1406/B 4000 x 25 μl rxns

MASTER MIX

Master Mix containing DNA Polymerase, MgCl2 and dNTPs

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DNA Polymerase /10X ReddyMix PCR Buffer / MgCl2

REDDYMIX MASTER MIX


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DNA Polymerase / 10X ReddyMix PCR Buffer / MgCl2 / 20 mM dNTPs

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Savetime with ReddyMix! Go straight from PCR to gel loading with ReddyMix Formats


MgCl2 Concentrations:All buffers pre-mixed with MgCl2 will be at 1.5 mM concentration in the final reaction. Alternative pre-mixed buffers are available containing MgCl2 concentrations of 2.0, 2.5, 3.0, 3.5, 4.0 mM. Please enquire.

KCl Buffer Options: All mixes contain (NH4)2 SO4 buffer. KCl based buffers are available upon request.

Master Mix Concentrations:2X Concentration allows for the addition of more template volume to accommodate dilute or low copy number templates (1.1X allows for of up to 1.5 ul of template, 2X allows for up to 11.5 ul of template).

Detailed Product InformationFor more information or to download the product manual, please visit: www.thermo.com/thermoprime



Hot-Start PCRThermo-Start Taq DNA PolymeraseDuring PCR set-up, reaction samples may be exposed to sub-optimal annealing temperatures, allowing primers to anneal to each other or to non-specific segments of the template. During this time, active DNA polymerase can extend these misaligned primers to create primer-dimers or non-specific product. This amplification of non-specific product continues throughout PCR cycling, consuming reaction components and reducing the amplification efficiency for the target sequence, resulting in reduced assay sensitivity and end yield of the target product.

Thermo-Start Taq DNA Polymerase eliminates this non-specific amplification by preventing DNA polymerase activity from occurring during assay set-up. The polymerase activity is suspended using a covalent chemical modification that inhibits all enzyme activity. This modification is maintained until reversed by a 15 minute 95oC activation step. In addition, the Thermo-Start buffering system contains heat stabilizing components that protect Thermo-Start over the multiple high temperature steps of PCR cycling, to deliver highly efficient, specific amplification of even very low copy number targets. A high performance buffer is also available for GC-rich targets or other challenging templates with a high degree of secondary structure.

Cat. No. Pack Size

AB-0908/A 400 x 25 μl rxns

AB-0908/B 4000 x 25 μl rxns


AB-1908/A 400 x 25 μl rxns

AB-1908/B 4000 x 25 μl rxns

w/ High Performance Buffer Formulation

AB-1057/A 400 x 25 μl rxns

AB-1057/B 4000 x 25 μl rxns

Cat. No. Pack Size

AB-0908/A/N 400 x 25 μl rxns

AB-0908/B/N 4000 x 25 μl rxns


AB-1908/A/N 400 x 25 μl rxns

AB-1908/B/N 4000 x 25 μl rxns


AB-1057/A/N 400 x 25 μl rxns

AB-1057/B/N 4000 x 25 μl rxns

Cat. No. Pack Size

1.1X Concentration

AB-0938/15/A 160 x 25 µl rxns

AB-0938/15/B 1600 x 25 µl rxns

2X Concentration

AB-0938/15/DC/A 160 x 25 µl rxns

AB-0938/15/DC/B 1600 x 25 µl rxns


2X Concentration

AB-1147/A 160 x 25 µl rxns

AB-1147/B 1600 x 25 µl rxns

Cat. No. Pack Size

AB-1950/A 400 x 25 μl rxns

AB-1950/B 4000 x 25 μl rxns


AB-1951/A 400 x 25 μl rxns

AB-1951/B 4000 x 25 μl rxns

Cat. No. Pack Size

1.1X Concentration

AB-1960/A 160 x 25 µl rxns

AB-1960/B 1600 x 25 µl rxns

2X Concentration

AB-1961/A 160 x 25 µl rxns

AB-1961/B 1600 x 25 µl rxns


2X Concentration

AB-1962/A 160 x 25 µl rxns

AB-1962/B 1600 x 25 µl rxns

Cat. No. Pack Size

AB-1950/A/N 400 x 25 μl rxns

AB-1950/B/N 4000 x 25 μl rxns


AB-1951/A/N 400 x 25 μl rxns

AB-1951/B/N 4000 x 25 μl rxns

Benefits:

• Hot-Start activation for stringent specificity

• Hot-Start modification allows for room temperature set-up

• Enhanced enzyme stability for increased yield

• High Performance Buffer for GC-rich templates

THER

MO

-STA

RT

PCR amplification of a 250 bp and 630 bp β-actin gene fragment from 3 concentrations (100, 50 and 25 ng) of human genomic DNA using either Thermo-Start Taq or an equivalent hot-start Taq from Supplier A, Q or I.

Figure 5: Thermo-Start Taq DNA Polymerase exhibits enhanced specificity and sensitivity over other hot-start enzymes.

100 50 25

Supplier I

250 bp >>

Thermo-Start Supplier A Supplier QGenomic DNATemplate (ng) >> 100 50 25100 50 25100 50 25

630 bp >>

MgCl2 Concentrations:All buffers pre-mixed with MgCl2 will be at 1.5 mM concentration in the final reaction. Alternative pre-mixed buffers are available containing MgCl2 concentrations of 2.0, 2.5, 3.0, 3.5, 4.0 mM. Please enquire.

Master Mix Concentrations:2X Concentration allows for the addition of more template to accommodate dilute or low copy number templates. (1.1X allows for of up to 1.5 ul of template, 2X allows for up to 11.5 ul of template.)

Detailed Product InformationFor more information or to download the product manual, please visitwww.thermo.com/thermostart

95

72

60

20PCR Protocol Steps

InitialDenaturation

Primer Annealing

Potential for Non-specific amplification

Primer Extension

CyclingDenaturation

Sub-optimal Annealing TemperatureTem

pera

ture

(ºC)

Thermo-Start activation

Figure 4: Thermo-Start Taq DNA Polymerase remains completely inactive until the 95oC activation step.

PCR samples are subject to sub-optimal annealing conditions during PCR set-up and transport, as well as during the initial denaturation cycle. It is important that the DNA polymerase remains inactive until conditions are optimal for specific amplification.

High Performance Buffer is recommended exclusively for GC-rich or other

complex targets


MASTER MIX

Master Mix containing DNA Polymerase, MgCl2 and dNTPs

ENZYME KIT


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DNA Polymerase /10X ReddyMix PCR Buffer / MgCl2

REDDYMIX MASTER MIX


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DNA Polymerase / 10X ReddyMix PCR Buffer / MgCl2 / 20 mM dNTPs

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PCR Plastics

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High Fidelity PCRVerbatim High Fidelity DNA PolymeraseDemanding applications, such as cloning and mutagenesis, require a high performance proofreading enzyme that delivers maximum accuracy and yield. Standard proofreading enzymes such as Pfu or KOD have only moderate fidelity and exhibit reduced processivity due to their proofreading activity. This results in a low yield PCR product that contains an increased number of sequence errors.

Thermo Scientific Verbatim High-Fidelity DNA Polymerase is an advanced proofreading enzyme that delivers superior fidelity and processivity. The Verbatim enzyme is a modified DNA polymerase with enhanced proofreading activity and high template binding affinity. The enhanced proofreading activity generates highly accurate PCR products, and the increased affinity dramatically improves processivity and yield. Additionally, the increased processivity allows for up to 75% reduction in PCR protocol times for faster results.

The high template binding affinity also helps Verbatim work through difficult templates, making it a robust enzyme successful on a wide variety of targets. Verbatim High-Fidelity DNA Polymerase is provided with two optimized buffering systems, our high-fidelity buffer for success with most templates, and a GC buffer for success with GC-rich and other difficult to amplify templates.

Cat. No. Pack Size

AB-1920/A 200 x 25 µl rxns

AB-1920/B 1000 x 25 µl rxns

Cat. No. Pack Size

AB-1920/A/N 200 x 25 µl rxns

AB-1920/B/N 1000 x 25µl rxns

Benefits:

• Superior proofreading capabilities for industry leading fidelity

• Enhanced processivity for increased yield and up to 75% reduction in protocol times

• Flexible buffering systems for success with a variety of templatesi

Choose Your Preferred Format:Figure 6: Verbatim High Fidelity DNA Polymerase exhibits superior fidelity compared to Taq, standard proofreaders, and advanced fusion proofreading enzymes.

Average error rates of five PCR enzymes, including Verbatim High Fidelity DNA Polymerase (data generated following a modified protocol based on the rpsL method described by Fuji et al, 1999).

Notes

MgCl2 Concentrations:Both the High Fidelity and GC buffers are pre-mixed with MgCl2 at 2.0 mM concentration in the final reaction. The separate MgCl2 vial can be used for further MgCl2 optimization.

High Fidelity Buffer and GC Buffer:Use High Fidelity Buffer for all standard templates. Use GC Buffer only for GC rich or other complex templates.

T/A Cloning:Due to its proof-reading activity, Verbatim removes the 3’-A overhangs necessary for T/A cloning applications. Protocols are readily available for the addition of 3’-A overhangs post-amplification. Please contact our technical support team for more information.

Detailed Product InformationFor more information or to download the product manual, please visit www.thermo.com/verbatim

Figure 8: Shortened extension times allow for up to 75% reduction in overall protocol times.

Calculated PCR amplification protocol times using Pfu, Taq or Verbatim with 1, 2.8, and 5 kb templates. Protocol times are based on suppliers recommended 3-step reaction protocol repeated over 30 cycles.

Enzyme Fidelity

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

1.40E+05

1.60E+05

Taq Pfu/Taq Blend KOD Leading FusionEnzyme

Verbatim

(1/ A

vera

ge

Erro

r R

ate)

Figure 7: Verbatim’s high affinity to DNA templates increases processivity to deliver improved yield over standard Taq and Pfu.

PCR amplification of pKF3 plasmid (2.3 kb) using either standard Taq, a Pfu enzyme, or Verbatim with extension times of either 15 sec, 30 sec or 60 sec, during a 40 cycle protocol. All products are run with a 1 kb ladder (AB-0387).

2.8 kb >>

15 sec/ kb

Taq

Ver

Pfu

Taq

Ver

Pfu

Taq

Ver

Pfu

30 sec/ kb 60 sec/ kbV

ER

BA

TIM


kb

5kb Not recommended

6 hours

1 hour, 35 min

1kb

2 hours

1 hour

35 min

2.8kb

PFU

Taq

Verbatim

3 hours, 45 min

2 hours

1 hourTem

plat

e Le

ngth

Total Protocol Time

DNA Polymerase / 5X High Fidelity Buffer / 5X GC Buffer / MgCl2

ENZYME KIT

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COMPLETE PCR KIT

DNA Polymerase / 5X High FidelityBuffer / 5X GC Buffer / MgCl2 / dNTPs

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PCR Plastics

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Control Template & Primer Formats:These PCR kits include control genomic DNA with two sets of primers designed to create 5 kb and 15 kb fragments. Intended for use as a positive control in long range PCR.

Buffer 1 and Buffer 2:Use Buffer 1 for target sequences up to 12 kb. Use Buffer 2 for targets 12 - 20 kb, GC-rich or problematic targets of any length.

MgCl2 Concentrations:All buffers pre-mixed with MgCl2 will be at 1.5 mM concentration in the final reaction.

Master Mix Concentrations:2X Concentration allows for the addition of more template volume to accommodate dilute or low copy number templates.

Detailed Product Information:For more information or to download the product manual, please visit www.thermo.com/extensor

Notes



Cat. No. Pack Size

AB-0720/A 80 x 25 μl rxns

AB-0720/B 400 x 25 μl rxns

w/ Control Template & Primers


AB-0721/B 400 x 25 μl rxns

Cat. No. Pack Size

AB-0720/A/N 80 x 25 μl rxns

AB-0720/B/N 400 x 25 μl rxns

w/ Control Template & Primers

AB-0721/A/N 80 x 25 μl rxns

AB-0721/B/N 400 x 25 μl rxns

Cat. No. Pack Size

w/ Buffer 1 Formulation (2X Conc.)

AB-0792/A 80 x 25 µl rxns

AB-0792/B 400 x 25 µl rxns



AB-0793/B 400 x 25 µl rxns

Cat. No. Pack Size



AB-0794/B 400 x 25 µl rxns



AB-0795/B 400 x 25 µl rxns

Benefits:

• Proprietary enzyme blend amplifies targets up to 20 kb in length

• Unique blend and optimized buffer combination delivers increased accuracy and yield

• Alternative buffering system for success with GC-rich targets

REDDYMIX MASTER MIX

ReddyMix Master Mix containing Polymerase Blend, MgCl2 and dNTPs

PCR amplification of 2, 5, 7, 9, 11, 13 and 15 kb fragments of the human tissue plasminogen activator gene from human genomic DNA and a 20 kb fragment from lambda DNA.

Figure 9: The two enzymes in the Extensor blend act synergistically to generate long length PCR products.

Figure 10: The Extensor enzyme blend delivers robust amplification of long range targets.

2 5 7 9 11 13 15 20Genomic DNATarget ( kb) >>

MASTER MIX

Master Mix containing Polymerase Blend, MgCl2 and dNTPs

Polymerase Blend / 10X PCRBuffer 1 / 10X PCR Buffer 2 / MgCl2

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Polymerase Blend / 10X Buffer 1 / 10X Buffer 2 / MgCl2 / 20 mM dNTPs

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Long Range PCRExtensor Long Range Enzyme BlendWhen working with longer length targets, it is important to use an appropriate enzyme system to ensure the generation of full length product. Standard Taq DNA polymerase incorporates approximately 1 error in every 9 kb. This error can cause the polymerase reaction to stop, or proceed very slowly, leading to incomplete amplicons. For long PCR, a system composed of a DNA polymerase and a proofreading enzyme, will give successful amplification; the DNA polymerase performs the nucleotide additions, and the proofreading enzyme corrects any mismatched bases, allowing the polymerase to amplify long sections of the template.

Extensor Long Range Enzyme Blend is an optimized mix of ThermoPrime DNA Polymerase and a proprietary thermostable proofreading enzyme. The two enzymes act synergistically to generate long PCR products, up to 20 kb. This makes the enzyme blend ideal for robust full-length amplification of long range sequences. PCR products generated with Extensor can be used directly in T/A cloning. Despite the 3’ to 5’ exonuclease (proofreading) activity present in the mix, the ThermoPrime Taq DNA Polymerase generates sufficient product with an A overhang, eliminating the need to add A overhangs post PCR. Extensor Long Range Enzyme Blend is available with 2 buffer formulations; Buffer 1 for success with most templates up to 12 kb in length, and Buffer 2 for templates 12 - 20 kb and GC-rich or other difficult templates.


Control Template & Primer Formats include genomic DNA with forward and reverse primers


Available


PCR Plastics

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2-Step RT-PCR:

RT-PCRVerso Reverse TranscriptaseSuccessful RT-PCR analysis depends greatly on a robust and sensitive reverse transcription reaction; one with a processive RT enzyme that can effectively transcribe through difficult RNA secondary structure, and a priming method that ensures high cDNA yield while maintaining an unbiased representation of the RNA population.

The Verso system achieves robust and sensitive reverse transcription through the combination of a high affinity RT enzyme, a unique RNA priming method, and an optimized buffering system. The Verso RT enzyme has high RNA template affinity and reduced RNase H activity, allowing the enzyme to generate long cDNA fragments. The generation of long, uninterrupted fragments demonstrates that even sections with high secondary structure are successfully reverse transcribed, ensuring that all RNA sequences are represented in the cDNA pool (Figure 12). The included anchored oligo dT priming method further enhances sensitivity by increasing transcription efficiency (Figure 11).

There are two buffering options for the Verso enzyme; a cDNA synthesis buffer, and a 1-Step RT-PCR buffer. The cDNA synthesis buffer has been optimized to achieve a full and diverse cDNA pool, and the 1-Step system has been developed to attain high performance from both the reverse transcriptase and the DNA polymerase, delivering superior sensitivity and yield.

Cat. No. Pack Size


AB-1453/B 100 x 20 μl rxns

PCR Kit Page

Standard PCR 7

High Yield PCR 8

Hot-Start PCR 10

High Fidelity PCR 12

Long Range PCR 14

Benefits:

• High affinity RT enzyme for robust cDNA synthesis

• Efficient RT reaction allows for 75% shorter protocol times (down to 30 minutes)

• Unique priming strategy for broad template success

• Wide working temperature range for GC rich targets

• RT Enhancer eliminates the need for DNase treatment

Figure 13: RT Enhancer degrades genomic DNA, eliminating the need for separate DNase treatment.

The RT Enhancer, which is included in all Verso kits, is a unique enzyme that degrades contaminating genomic DNA at the start of the RT step. It is equally as effective as DNase I treatment but more convenient as it eliminates the need for a separate DNase I incubation prior to RT-PCR. Figure below demonstrates equal DNA degradation with RT enhancer and DNase I treatment.

Figure 12: Verso exhibits high affinity for mRNA templates giving long and high-yield cDNA providing an accurate cDNA pool for PCR.

Following reverse transcription using the Verso cDNA Synthesis Kit, PCR primers pairs were used to amplify short fragments 0.56 kb, 9.7 kb and 11.1 kb from the mRNA / poly(A) junction. The successful amplification of all 3 amplicons, even from very low input concentrations, demonstrates that Verso RT has a high affinity for RNA and can reverse transcribe long RNA templates. NTC – no template control; NEC – no enzyme control.

10ng 1ng 100pg NTC NECM 100ng

cDN

A le

ngth

0.56 kb

9.7 kb

11.1 kb

Pair with one of our PCR kits for complete 2-Step RT-PCR

cDNA SYNTHESIS KIT

RT Enzyme / 5X RT Buffer /Random Hexamers / Anchored Oligo

dT / 20 mM dNTPs / RT Enhancer

TTTTTTTTTT3’ 5’

Standard oligo dT

AAAAAAAAAAAAAAAmRNA5’ 3’

TTTTTTTTTT3’ 5’

AAAAAAAAAAAAAAAmRNA5’ 3’

Anchored oligo dT

N V

V dGTP, dATP or dCTP

any base

Figure 11: Utilizing Anchored oligo dT in place of standard oligo dT increases reverse transcription efficiency.

Standard oligo dT anneals anywhere along the poly A tail, and can therefore prime internal sites within the poly A tail or other poly A rich regions.

Anchored oligo dT anneals only at the mRNA / poly A tail junction, preventing inefficient “inner A tail” priming and increasing mRNA transcription efficiency.

RT

EN

ZYM

E

BU

FFE

R

PR

IME

R 2

dNTP

s

RT

EN

H.

PR

IME

R 1

1-Step RT-PCR:

Cat. No. Pack Size

w/ ThermoPrime Taq DNA Polymerase


AB-1454/B 400 x 25 µl rxns

Hot-Start Version

w/ Thermo-Start Taq DNA Polymerase


AB-1455/B 400 x 25 µl rxns

Cat. No. Pack Size

w/ ThermoPrime Taq DNA Polymerase

AB-1454/LD/A 80 x 25 μl rxns

AB-1454/LD/B 400 x 25 μl rxns

1-STEP RT-PCR MASTER MIX

RT Enzyme / 2X Master Mix Buffer containing DNA Polymerase, MgCl2

and dNTPs / RT Enhancer

RT

EN

ZYM

E

BU

FFE

R

RT

EN

H.

1-STEP RT-PCR REDDYMIX MASTER MIX

RT Enzyme / 2X Master Mix ReddyMix Buffer containing DNA Polymerase,

MgCl2 and dNTPs / RT Enhancer

RT

EN

H.

BU

FFE

R

RT

EN

ZYM

E

VE

RS

OP

CR

RT Controls:

Cat. No. Pack Size


RNA Control Kit

The RNA control kit is intended for use as a positive control to monitor the performance of all

RT PCR components. The kit includes a 3.6 kb RNA synthesized according to the sequence

derived from bacteriophage MS2 and primers designed to generate a 522 bp product.

RT

RNA CONTROL KIT

Positive Control RNA / Control Primer 1 / Control Primer 2

TEM

PLA

TE

PR

IME

R 1

PR

IME

R 2


M 1 2 3 4 M Genomic DNA treated with:

1 (No treatment) 2 RT Enhancer 3 (No treatment) 4 DNase treatment

M = DNA Marker


For

more sealing

options, please refer

to our full PCR Plastics guide.

More

options available,

please refer to our full PCR

Plastics guide.


PCR Plastics


ABgene PCR Tubes, Plates and Seals High quality PCR plastics are an important factor in achieving sensitive and accurate PCR results. The wells must deliver fast, uniform heat transfer for maxi mum and consistent PCR efficiency. Plates and seals must be engineered to work together to prote ct samples from evaporation, and the plastics must be manufactured in a process that avoids impurities and contaminants that can affect molecular biology applications.

Thermo Scientific ABgene PCR plastics are cleanroom produced and expertly manufactured to ensure maximum PCR performance. To create a highly inert well surface, all plastics are made from virgin medical grade polypropylene, and molding tools are maintained without the use of lubricating agents. Molds are precision designed and highly polished to create uniform, ultra thin, smooth well walls.

The quick order tables below represent some of the most popular formats, but the full range of formats and product options is available online and in our PCR plastics selection guide. (See below for details)

Popular Formats:

Popular Formats:

Popular Formats:

Cat. No. Pack Size

Clear

AB-0337 1000 Tubes

Assorted Colors

AB-0491 1000 Tubes

Cat. No. Pack Size

Clear

AB-0600 25 Plates

Assorted Colors

AB-0600/B 25 Plates

AB-0600/G 25 Plates

AB-0600/P 25 Plates

AB-0600/R 25 Plates

AB-0600/Y 25 Plates

Cat. No. Pack Size

AB-0626 100 Seals

Cat. No. Pack Size

Clear

AB-0620 1000 Tubes

Assorted Colors

AB-0622 1000 Tubes

Cat. No. Pack Size

Clear

AB-1400 25 Plates

Clear with Barcodes

BC-1400 25 Plates

Cat. No. Pack Size

AB-0558 100 Seals

Cat. No. Pack Size

Domed Caps (individually attached)

AB-1504 250 Strips

Flat Caps (individually attached)

AB-1502 250 Strips

Cat. No. Pack Size

Clear

AB-2150 50 Plates

Flat Caps

BC-3150 50 Plates

Cat. No. Pack Size

Domed

AB-0265 250 Strips

Flat

AB-0784 250 Strips

Compatible with most 0.2 mL cycler blocks Compatible with most 0.2 mL cycler blocks

Compatible with most 0.2 mL 96-Well cycler blocks.

For a full list of compatible instruments, visit www.thermo.com/pcrplastics

Compatible with most 384-Well cycler blocks.

Compatible with most 0.2 mL 96-Well cycler blocks.

Compatible with most 0.2 mL cycler blocks

0.2 ML DOMED CAP PCR TUBES

96-WELL PCR PLATE, NON-SKIRTED

ADHESIVE PCR FOIL SEAL0.2 ML FLAT CAP PCR TUBES

96-WELL PCR PLATE, SEMI-SKIRTED

ADHESIVE PCR FILM SEAL0.2 ML EASYSTRIP PCR TUBES

384-WELL DIAMOND ULTRA PCR PLATE

8-STRIP PCR CAP STRIPS

TU

BE

S

PLA

TE

SS

EA

LS

Benefits:

• Cleanroom produced for contamination-free PCR

• Uniform, ultra-thin well walls for maximum efficiency and reproducibility

• Plates and seals are precision designed to minimize evaporation

• RNase, DNase, Endotoxin, and Human DNA free

To view the complete range of Thermo Scientific ABgene plastics or to order our

PCR Plastics Selection Guide, please visit: www.thermo.com/pcrplastics

Endo-toxins

RNaseDNase

HumanDNA

To

view our complete range

of compatibleplates, please

refer to our full PCR Plastics

guide.

0420 www.thermo.com/pcrsimplified 210420 www.thermo.com/pcrsimplified 21

Custom Aliquoting ServicesCustom Solutions for High-Throughput PCRThermo Scientific Custom Aliquoting Services can significantly streamline your process and increase your PCR productivity. Our Custom Aliquoting Service can provide any of our PCR and qPCR mixes pre-aliquoted into our high quality PCR plastics. Save time, money, and effort by letting our PCR focused manufacturing facility, state-of-the-art instruments, and skilled production staff supply you with reliable, quality controlled, pre-filled PCR plastics.

PCR Plastic Format OptionsThermo Scientific ABgene PCR plastics are expertly designed and cleanroom produced for the ultimate in PCR reliability. All products are manufactured under strict ISO 9001 guidelines and go through extensive QC testing to ensure PCR performance. Our quality PCR plastics are available in a variety of formats to fit any cycler and suit any workflow.

Let Thermo Scientific Custom Aliquoting Services Streamline Your PCR Workflow

Simplify Supply Chain & Materials ManagementBuying pre-filled PCR plastics greatly simplifies the procurement process. All reagent and plastic components are ordered with a single part number and can be purchased in a single transaction. Standing orders can be set up to deliver regular shipments to match production demand. The supply chain is simplified by having one vendor for all PCR consumables, and stock management is far easier when the products arrive in a pre-assembled format.

Improve Quality Control and DocumentationStop collecting and managing QC documentation from multiple suppliers, for multiple products and lot numbers. All of our pre-aliquoted products are manufactured under strict ISO 9000 quality control guidelines and are supplied with certificates of analysis for each reagent and plastics lot. We can also provide entire orders consisting of a single combination of plastic and reagent lot numbers for easy QC tracking. End the struggle to manage QC data and increase assay reproducibility by switching to our pre-filled PCR plastics.

Reduce Assay Failure and Increase ProductivityIncrease productivity by letting us take care of your reaction mix and aliquoting steps. In-house master mix creation and aliquoting can introduce multiple variables in your production process. Don’t waste valuable time and resources troubleshooting through multiple production steps. Let our 24/7 PCR manufacturing facility provide you with an uninterrupted supply of cycler-ready, reliable product to keep your project moving.

In-House HTP Assay Set-upand Management

Multiple SuppliersFor every separate product

that is purchased, there are separate orders, invoices,

and stocking. Procurement and management of these

components is a significant investment of time

and resources.

Multiple In-house Set-up Steps

Having multiple in-house set-up steps introduces

variables into your production process. This increases the

likelihood of failure, and makes troubleshooting a long and

difficult process.

Multiple QC/QADocumentsEach separate component has its own QC documentation, and multiple lot numbers for each component add to the complexity of QC management.

DISPENSEMASTER MIX

ADD TEMPLATEAND PRIMERS

RUN PCR &ANALYZE RESULTS

IN-HOUSE PRODUCTIONOF MASTER MIX

Thermo Scientific Custom Aliquoting Services

Single SupplierPlastics and reagents are all from the same supplier, simplifying ordering and materials maintenance and storage.

Minimal In-house Set-up StepsMost in-house set-up steps are eliminated with pre-filled plastics. Our PCR focused manufacturing facilityoperates under ISO 9001 guidelines to create, assemble and deliver production quality pre-filled plates.

Simplified QC/QA Documentation

Produced under strict QC guidelines and can

be supplied with QC documentation for reagent and plastic. Orders can be

made up of single lot numbers for reagent and plastic.

ADD TEMPLATEAND PRIMERS

RUN PCR &ANALYZE RESULTS

To learn more about our custom aliquoting solutions, please visit:

www.thermo.com/custompcr

STRIP TUBESINDIVIDUAL TUBES

96-WELL PLATES 384-WELL PLATES

VS


Ordering Information

United StatesFisher Scientific: (All Products) Direct: (PCR Reagents Only)

Telephone: (800) 766-7000 Telephone (800) 874-3723

To fax an order: (800) 926-1166 To fax an order (800) 842-5007

Order online: www.fishersci.com Product Information: www.thermo.com/pcrproducts

United KingdomDirect: (All Products)

Telephone: 01372 723456

To fax an order: 01372 741414

Order online: www.thermo.com/abgene

FranceDirect: (PCR Reagents Only) Dominique Dutscher: (All Products) Fisher Scientific: (All Products)

Telephone: 01 60 92 48 00 Telephone: 03 88 59 33 90 Telephone: 03 88 67 53 23

To fax an order: 01 60 92 49 00 To fax an order: 03 88 59 33 99 To fax an order: 03 88 67 85 11

Order online: www.thermo.com/abgene Order online: www.dutscher.com Order online: www.fr.fishersci.com

GermanyDirect: (All Products)

Telephone: 040 23 00 21

To fax an order: 040 23 00 55

Order online: www.thermo.com/abgene

Rest of World Please refer to our distributor listing by visiting www.thermo.com/PCRcontact

Need help? Contact your local technical support team

United StatesTelephone: (800) 235-9880, option #4 [email protected]

United KingdomTelephone: 01372 840 410 [email protected]

France

Telephone: 01 60 92 48 68 [email protected]

GermanyTelephone: 040 23 51 36 79 [email protected]

Rest of World Telephone: +44 1372 840 410 [email protected]

0424

Thermo Scientific PCR Reagent Selection Guide

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