PCR REAGENTS PCR & RT-PCRbiology.hunter.cuny.edu/molecularbio/Class... · (2) Use Multiplex PCR 5X Master Mix. (3) ™Use Quick-Load Taq 2X Master Mix. (4) ™Use Crimson LongAmp

CLONING & MAPPING

DNA AMPLIFICATION & PCR

RNA ANALYSIS

PROTEIN EXPRESSION & ANALYSIS

GENE EXPRESSION & CELLULAR ANALYSIS

PCR REAGENTS PCR & RT-PCR

Phusion®* Phusion®

Hot Start II* Phusion®

Flash* TaqLongAmp™

Taq Crimson Taq VentR® Deep VentR

®

Fidelity vs. Taq 50X 50X 25X 1X 2X 1X 5X 6X

Amplicon Size ≤20 kb ≤20 kb ≤20 kb ≤5 kb ≤30 kb ≤5 kb ≤6 kb ≤6 kb

Extension Rate 2-4 kb/min 2-4 kb/min 4 kb/min 1 kb/min 1.2 kb/min 1 kb/min 1 kb/min 1 kb/min

Resulting Ends Blunt Blunt Blunt 3´ A 3´ A/Blunt 3´ A Blunt Blunt

3´→5´ exo Yes Yes Yes No Yes No Yes Yes

5´→3´ exo No No No Yes Yes Yes No No

Units/50 µl Reaction 1.0 1.0 N/A 1.25 5.0 1.25 0.5–1.0 0.5–1.0

Routine PCR l l l l l l l l

High Fidelity l l l l l l

High Yield l l l l l l

Hot Start l l

Fast l l l

Long Amplicon l l l l

Difficult (GC-rich) Templates l l l l l

High Throughput l l l l l

Multiplex PCR l(2)

Extraction-free PCR

DNA-labeling l

Site-directed Mutagenesis l(1)

Kit l l l

Master Mix l l l l

Direct Gel Loading l(3) l(4) l

2

PCR Polymerase Selection Chart

(1) Use Phusion® Site Directed Mutagenesis Kit.

(2) Use Multiplex PCR 5X Master Mix.

(3) Use Quick-Load™ Taq 2X Master Mix.

(4) Use Crimson LongAmp™ Taq DNA Polymerase.

* Produced by Finnzymes Oy. Distributed by New England Biolabs, Inc.

For over 35 years, New England Biolabs, Inc. has been a world leader in the discovery and production of reagents for the life science community. NEB offers one of the largest selections of polymerases for PCR applications and through our commitment to research, ensures the development of innovative tools for PCR. Our product quality, enzyme expertise and outstanding technical support bring unparalled confidence to your PCR experiments.

CLONING & MAPPING

DNA AMPLIFICATION & PCR

RNA ANALYSIS

PROTEIN EXPRESSION & ANALYSIS

GENE EXPRESSION & CELLULAR ANALYSIS

PCR REAGENTS FROM NEB P

RO

PE

RT

IES

AP

PLI

CA

TIO

NS

FOR

MA

TS

www.neb.com

DyNazyme™ II

Hot Start* DyNAzyme™

Ext*

Phire® Hot Start

II*Hemo

KlenTaq™

Phusion® Blood Direct*

Phire® Animal Tissue Direct

PCR Kit*

Phire® Plant Direct PCR

Kit*

Fidelity vs. Taq 1X 2X 2X ND 25X 2X 2X

Amplicon Size ≤3 kb ≤40 kb ≤7.5 kb ≤2 kb ≤7.5 kb ≤7.5 kb ≤7.5 kb

Extension Rate 1 kb/min 1.3-1.5 kb/min 3 kb/min 0.5 kb/min 2-4 kb/min 3 kb/min 3 kb/min

Resulting Ends 3´ A 3´ A/Blunt Blunt 3´ A Blunt Blunt Blunt

3´→5´ exo No Yes Yes No Yes Yes Yes

5´→3´ exo No No No No No No No

Units/50 µl Reaction 0.75–2.0 0.5–3.0 N/A N/A N/A N/A N/A

Routine PCR l l

High Fidelity l

High Yield l l l l l l

Hot Start l l l l l

Fast l l l l

Long Amplicon l

Difficult (GC-rich) Templates l

High Throughput l l

Multiplex PCR l

Extraction-free PCR l l l l

DNA-labeling

Site-directed Mutagenesis

Kit l l l l

Master Mix

Direct Gel Loading

3

SELECTION CHART

* Produced by Finnzymes Oy. Distributed by New England Biolabs, Inc.

N/A Not applicable

PR

OP

ER

TIE

SA

PP

LIC

AT

ION

SFO

RM

AT

S

Visit confidentPCR.com to learn more,

and to find PCR-related special offers.

Phusion® High-Fidelity DNA Polymerase

The unique Phusion® High-Fidelity DNA Polymerase offers robust performance. Phusion DNA Polymerase generates products with accuracy and speed previously unattainable with a single enzyme, even on the most difficult templates. Its unique structure, a novel Pyrococcus-like enzyme fused with a processivity-enhancing domain, enhances fidelity, making Phusion DNA Polymerase a superior choice for cloning and setting a new standard for PCR performance. With an error rate 50-fold lower than that of Taq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus DNA Polymerase, it is the most accurate thermostable polymerase available. Additionally, Phusion DNA Polymerase possesses processivity 10-fold greater than Pyrococcus furiosus DNA Polymerase and twice that of Taq DNA Polymerase.

Phusion® High-Fidelity DNA Polymerase ................................................................................ F-530S/L

Phusion® High-Fidelity PCR Kit ............................................................................................... F-553S/L

Phusion® High-Fidelity PCR Master Mix with HF Buffer ..................................................... F-531S/L

Phusion® High-Fidelity PCR Master Mix with GC Buffer .................................................... F-532S/L

4

Advantages:• Extreme Fidelity - Highest of any

commercially available thermostable polymerase (50X greater than Taq )

• High Speed - Extension times are dramatically reduced (10X faster than Pfu )

• Robust Reactions - Maximal success with minimal optimization

• High Yield - Increased product yield with minimal enzyme amount

• Specificity - Hot-start modification reduces non-specific amplification and primer degradation

A 3.8 kb fragment from human beta globin gene was amplified according to suppliers’ recommendations using varying extension times. One unit of Phusion DNA Polymerase amplified the fragment with a combined annealing and extension step of only 1 minute, with higher yields than 2.5 or 5 units of Pyrococcus furiosus DNA Polymerases.

Pyrococcus modified Phusion DNA furiosus P. furiosus Polymerase (5 units) (2.5 units) (1 unit)

Experience extreme speed and yield with Phusion DNA Polymerase

min 1 1.5 3.8 7.6 1 1.5 3.8 7.6 1 1.5 3.8 7.6

PHUSION DNA POLYMERASE

Phusion Buffer Selection Chart

CHOICE OF BUFFER PRESENCE OF DETERGENT NEB #

Phusion HF Bufferdefault buffer for high-fidelity amplification

Phusion HF Buffer Pack F-518S/L

Detergent-free Phusion HF Buffer Pack F-520S/L

Phusion GC Bufferfor long, difficult or GC-rich templates (when HF buffer fails)

Phusion GC Buffer Pack F-519S/L

Detergent-free Phusion GC Buffer Pack F-521S/L

Visit www.neb.com/phusion for more information.

High Fidelity High Yield Hot Start Fast PCR Long PCR Difficult Templates Convenience Extraction-free PCR

www.neb.com 5

Phusion® Flash enhances speed, yield and specificityThe fast reaction kinetics of Phusion® DNA Polymerase generates higher yields of PCR products using shorter reactions times. The Phusion Flash High-Fidelity PCR Master Mix takes this speed to even greater extremes, allowing extension times of only 15 seconds or less per kilobase. This polymerase utilizes a reversibly bound affibody which inhibits enzyme activity at ambient temperatures.

Phusion® Flash High-Fidelity PCR Master Mix ...................................................................... F-548S/L

Phusion® Hot Start offers extreme specificityThe Affibody®-based inactivation method of Phusion Hot Start II DNA Polymerase increases the specificity of PCR amplifications. Both the polymerase and the proofreading activity are inactive at room temperature, preventing non-specific amplification and primer degradation. This allows the reaction to be set up at room temperature, which is ideal for high-throughput technologies. In addition, Phusion Hot Start II DNA Polymerase delivers improved robustness, fewer reaction failures and minimal optimization.

Phusion® Hot Start II High-Fidelity DNA Polymerase ........................................................... F-549S/L

Tips for Using Phusion DNA Polymerase:• Use Phusion High-Fidelity DNA

Polymerase at 0.5–1.0 units per 50 µl reaction volume. Do not exceed 2 units/50 µl reaction.

• Use 15–30 sec/kb for extensions with Phusion DNA Polymerase. Do not exceed 1 min/kb.

• Use 200 µM dNTPs

• Do not use dUTP

• Use 98°C for denaturation

• Anneal at Tm+3°C (> 20 nt) or

use a 2-step protocol

• Phusion DNA Polymerase produces blunt end products

• Determine Tm using Finnzymes’

Tm calculator (www.finnzymes.com/

tm_determination)

A depiction of Phusion High-Fidelity DNA Polymerase. The double-stranded DNA binding domain (blue) is fused with a novel Pyrococcus-like enzyme (green) forming a unique high-performance polymerase.


Speed and robustness of the Phusion Flash High Fidelity PCR Master MixA 1.5 kb fragment from the human Cathepsin K gene was amplified with three different DNA polymerases according to the suppliers’ recommendations. Extension times were varied in a two-step protocol.

Comparison of performance versus extension times Pfu-based fusion Fast Taq Phusion Flash polymerase polymerase

Produced by Finnzymes Oy. Distributed by New England Biolabs, Inc.

Five proofreading DNA polymerases from major suppliers were used to amplify 1.7-2.3 kb amplicons from human genomic DNA. All amplifications were performed in accordance with manufacturers’ instructions. Phusion Hot Start II DNA Polymerase provided high yields of specific products whereas all other enzymes delivered lower or no yields, some of them also amplifying non-specific products.

Phusion Hot Start II provides extreme specificity and abundant yields

P: Phusion® Hot Start II High-Fidelity DNA PolymeraseA-D: Proofreading DNA polymerases from other leading suppliers

1.75 kb 2.2 kb 2.3 kb (GC-rich)M P A B C D M P A B C D M P A B C D M

6

Taq DNA Polymerase is the industry standard for routine PCR. NEB provides high quality recombinant Taq at an exceptional value. To accommodate a variety of PCR applications, Taq is available with different reaction buffers. Standard Taq Buffer is designed to support existing PCR platforms and is an ideal choice for DHPLC and high-throughput applications. ThermoPol Buffer is formulated to promote high product yields, even under demanding conditions.

Taq DNA Polymerase with ThermoPol Buffer .................................................................. M0267S/L/X

Taq DNA Polymerase with ThermoPol II (Mg-free) Buffer .................................................. M0321S/L

Taq DNA Polymerase with Standard Taq Buffer ............................................................... M0273S/L/X

Taq DNA Polymerase with Standard Taq (Mg-free) Buffer .................................................. M0320S/L

Taq PCR Kit ...................................................................................................................................... E5000S

Taq 5X Master Mix .................................................................................................................... M0285S/L

Taq 2X Master Mix .................................................................................................................... M0270S/L

Quick-Load™ Taq 2X Master Mix ............................................................................................ M0271S/L

Taq DNA Polymerase

Advantages:• Value - Industry standard for routine

PCR at a low price

• Versatility - Will amplify a wide range of templates with minimal optimization

• Flexibility - Able to incorporate dUTP, dITP and fluorescently-labeled nucleotides

• Choice - Reaction buffers accommodate a variety of PCR applications

• Convenience - Master mixes, buffer choice, tracking dye and kits available

Taq DNA POLYMERASE

Crimson Taq delivers additional convenienceExperience the robust and reliable performance of Taq DNA Polymerase in a more convenient format. Ideal for both routine and high throughput applications, Crimson Taq Reaction Buffer is optimized for robust amplification and contains tracking dye that allows samples to be loaded directly onto a gel. The Sampler contains sample sizes of Crimson Taq DNA Polymerase, dNTPs and the Quick-Load® 1 kb DNA Ladder at a value price.

Crimson Taq DNA Polymerase ................................................................................................. M0324S/L

Crimson Taq DNA Polymerase with (Mg-free) Buffer .......................................................... M0325S/L

Amplification of specific sequences from human genomic DNA using Crimson Taq DNA Polymerase. Amplicon sizes are indicated below gel. Marker M is NEB 1 kb DNA Ladder (NEB #N3232).

Taq Buffer Selection Chart

CHOICE OF BUFFER MG-CONTROL NEB #

Standard Taq Reaction Buffer:Detergent-free and designed to be compatible with existing assay systems

Taq with Standard Taq Buffer M0273

Taq with Standard Taq (Mg-free) Buffer M0320

ThermoPol Buffer:Designed to optimize yields and specificity

Taq with ThermoPol Buffer M0267

Taq with ThermoPol II (Mg-free) Buffer M0321

Crimson Taq Reaction Buffer:Contains crimson tracking dye, allowing samples to be loaded directly onto a gel

Crimson Taq M0324

Crimson Taq with (Mg-free) Buffer M0325

Amplification with Crimson Taq

Amplicon Size (kb)0.6 1.2 1.5 2.8 4.0 5.0 M

— 10.0

— 4.0

— 2.0

— 1.0

— 0.5

kb


Visit www.neb.com/taq for more information.

www.neb.com

LongAmp™ Taq enables extension of longer ampliconsAn optimized blend of Taq and Deep Vent

R® DNA Polymerases, LongAmp™ Taq DNA

Polymerase enables amplification of up to 30 kb PCR products with a fidelity higher than Taq DNA Polymerase alone. LongAmp Taq is also available with Crimson tracking dye in the reaction buffer.

LongAmp™ Taq DNA Polymerase ............................................................................................ M0323S/L

LongAmp™ Taq PCR Kit ................................................................................................................. E5200S

LongAmp™ Taq 2X Master Mix ............................................................................................... M0287S/L

Crimson LongAmp™ Taq DNA Polymerase ............................................................................ M0326S/L

Multiplex PCR 5X Master Mix for multiple templatesMultiplex PCR can simultaneously detect two or more products in a single reaction. Multiplex PCR can also be used for semi-quantitative gene expression analysis using cDNA templates. The NEB Multiplex PCR 5X Master Mix is an easy-to-use solution featuring high quality recombinant Taq DNA Polymerase. The mix is optimized for high yield and robust performance. Its performance is illustrated below in a 15-plex PCR reaction using human genomic DNA. The 5X formulation allows the user maximal flexibility for input of primers and template DNAs.

Multiplex PCR 5X Master Mix .................................................................................................... M0284S

7

Tips for Taq DNA Polymerase:• Use 20 pg–20 ng/ml of plasmid or viral

templates

• Use 20 ng–20 µg/ml of genomic templates

• Generally, primers should be 20–30 nucleotides in length, with an ideal GC content of 40–60%

• When engineering restriction sites into the end of primers, 4–6 extra bases should be added between the restriction site and the 5´ end of the primer

• Final primer concentration should be 0.1–0.5 µM each

• Final Mg2+ concentration should be 1.5–2.0 mM

• Typical dNTP concentration is 200 µM each

• Use 0.25–2.5 units of Taq/50 µl reaction

• Initial denaturation at 95°C is recommended

• Typical annealing temperatures are 5°C below the lowest T

m of the primers, and

are generally in the range of 55–60°C

• Extension at 68°C is optimal

Amplification of specific sequences from human genomic DNA using LongAmp Taq DNA Polymerase. Amplicon sizes are indicated below gel. Marker M is NEB 1 kb DNA Ladder (NEB #N3232).

Amplicon Size (kb)0.5 0.6 2 3 4 8 12 20 30 M

Amplification of longer templates with LongAmp Taq

kb

— 10.0

— 4.0

— 2.0

— 1.0

— 0.5

15-plex PCR reaction15-plex PCR using varying amounts of human genomic DNA. 1X Multiplex PCR 5X Master Mix was used with 0.15 μM of each primer. The cycling conditions were 95°C for 1 minute, 35 cycles of 95°C for 20 seconds, 60°C for 1 minute and 68°C for 2 minutes. Marker M is the 2-Log DNA Ladder (NEB #N3200).

kb— 0.8— 0.7— 0.6— 0.5

— 0.4

— 0.3

— 0.2

— 0.1

100 ng 10 ng 1 ng M


8

VentR

® DNA Polymerase

VentR

® DNA Polymerase is a recombinant, high-fidelity thermophilic DNA polymerase with the lowest cost per reaction of any high-fidelity PCR polymerase. It has an error rate 5-fold lower than Taq DNA Polymerase, a characteristic derived in part from an intrinsic 3´→5´ proofreading exonuclease. In addition, greater than 90% of the polymerase activity remains following a 1 hour incubation at 95°C, ensuring maximal activity over the course of the PCR reaction. For high-fidelity amplification of routine targets, Vent

R

DNA Polymerase is an exceptional value. VentR (exo-) DNA Polymerase has been

genetically engineered to eliminate the 3´→5´ proofreading exonuclease activity resulting in higher yield PCR.

VentR

® DNA Polymerase ............................................................................................................ M0254S/L

VentR

® (exo-) DNA Polymerase ................................................................................................. M0257S/L

Deep VentR

® DNA Polymerase is a recombinant, high-fidelity DNA polymerase with unsurpassed thermostability. This feature makes Deep Vent

R an ideal choice for PCR

amplification of DNA targets with a high degree of secondary structure, even in the absence of additives. It has an error rate 6-fold lower than Taq DNA Polymerase, a characteristic derived in part from an integral 3´→5´ proofreading exonuclease. Deep Vent

R’s combination of extreme thermostability and high-fidelity make it an excellent

choice for accurate PCR amplification of GC-rich sequences or templates with secondary structures. Deep Vent

R (exo-) DNA Polymerase has been genetically engineered to

eliminate the 3´→5´ proofreading exonuclease activity resulting in higher yield PCR.

Deep VentR

® DNA Polymerase .................................................................................................. M0258S/L

Deep VentR

® (exo-) DNA Polymerase ....................................................................................... M0259S/L

Deep VentR

® DNA Polymerase

Advantages:• High Fidelity - 5X greater than Taq

• High Thermostability - Half-life of 6.7 hrs. at 95°C

Advantages:• High Fidelity - 6X greater than Taq

• Extremely High Thermostability - Half-life of 23 hours at 95°C

• Difficult Templates - Ideal for GC-rich or looped sequences

VENT/DEEP VENT DNA POLYMERASE

Amplification with Vent and Deep Vent DNA PolymerasesAmplification of Jurkat Genomic DNA with VentR (A) and Deep VentR (B) DNA Polymerases. Amplicon sizes are indicated below the gel. Marker M is the 1 kb DNA Ladder (NEB #N3232).

Amplicon Size (kb)

0.7 1.1 2.0 M

A B

Amplicon Size (kb)

0.7 1.1 2.0 M


www.neb.com 9

DyNAzyme™ II Hot Start DNA Polymerase is a chemically modified form of the DyNAzyme II DNA Polymerase originating from Thermus brockianus. This modification renders it inactive until it undergoes a ten minute incubation at 94°C. This hot start feature prevents the formation of primer-dimers and extension of primers bound non-specifically during reaction set up.

DyNAzyme™ II Hot Start DNA Polymerase ............................................................................. F-504S/L

DyNAzyme™ II Hot Start DNA Polymerase

Advantages:• Specificity - Minimizes non-specific

products

• Convenience - Reactions can be set up at room temperature

• Flexibility - Able to incorporate dUTP and dITP

DYNAZYME DNA POLYMERASE


DyNAzyme™ EXT DNA Polymerase

DyNAzyme™ EXT DNA Polymerase is versatile, easy-to-use and especially suited for amplifying challenging templates without time-consuming optimization. It is an optimized mixture of two recombinant enzymes, DyNAzyme™ II DNA Polymerase and a proofreading enzyme.

DyNAzyme™ EXT DNA Polymerase ......................................................................................... F-505S/L

DyNAzyme™ EXT PCR Kit ........................................................................................................ F-552S/L

Advantages:• Length - Amplifies up to 40 kb

• Difficult Templates - Ideal for GC-rich or looped sequences

• Robust Reactions - High yield and robust reactions with standard templates

Amplification with DyNAzyme EXT DNA Polymerase yields highly specific products up to 40 kb in length. Reactions contained 2 ng of either an M13 (6.0 kb) or Lambda DNA template (0.5–2.6, 10–30 kb) and were conducted using supplied protocols. Amplicon sizes are shown below the gel. Marker (M) is Lambda DNA-Hind III Digest (NEB #N3012).

Long-range amplification by DyNAzyme EXT DNA Polymerase

M 0.5 2.6 6.0 10 15 20 25 30

Amplicon Size (kb)


Tips for Phire Hot Start II DNA Polymerase:• Use Phire Hot Start II DNA Polymerase

at 0.4 µl per 20 µl reaction and 1 µl per 50 µl reaction

• Use 20 sec/kb for extension

• Use 200 µM dNTPs

• Do not use dUTP

• Use 98°C for denaturation

• Anneal at Tm+3°C (> 20 nt) or use

2-step protocol

• Produces blunt end products

10

Phire® Hot Start II DNA Polymerase

Phire® Hot Start II DNA Polymerase outperforms Taq-based hot start DNA polymerases. This polymerase is significantly faster, extremely robust, and also capable of amplifying long DNA fragments with high yields. These features are achieved through advanced protein engineering of the polymerase. Phire Hot Start II DNA Polymerase incorporates a dsDNA-binding domain which allows short extension times (10-15 s/kb), improves yields, and increases fidelity 2-fold compared to Taq DNA polymerase. In addition, the unique hot start technology allows complete reactivation of the enzyme in “zero-time” at standard cycling temperatures. This combination of features makes the polymerase an ideal solution for routine and high throughput PCR applications.

Phire® Hot Start II DNA Polymerase ......................................................................................... F-122S/L

Advantages:• Fast - Amplification up to 4X faster than

hot start Taq DNA polymerases

• Robust Reactions - Maximal success with minimal optimization

• High Yield - Higher yields due to efficiency of polymerase

• Length - Amplification of products significantly longer than hot start Taq DNA polymerases

PHIRE DNA POLYMERASE

A 1.5 kb fragment from the human Cathepsin K gene was amplified with five different hot start DNA polymerases according to suppliers’ recommendations. Phire Hot Start II DNA Polymerase amplified high amounts of specific PCR product in just 29 minutes. In contrast, the PCR protocols for hot start Taq DNA polymerases from four major suppliers (A-D) were substantially longer and resulted in lower product yields.

A 600 bp fragment from human genomic DNA was amplified with hot start DNA polymerases from five major suppliers according to each supplier’s recommendations. Due to the unique hot start technology and a special dsDNA-binding domain in the Phire Hot Start II DNA Polymerase, the PCR protocol was completed up to four times faster than with Taq DNA polymerases utilizing chemically modified or antibody-based hot start technologies (suppliers A-D).

Phire Hot Start II Chemically Modified Antibody-based

M 29 Min 107 Min 106 Min 44 Min 97 Min

Abundant yields in shorter time with Phire Hot Start II

Complete PCR protocols in less than half the time using Phire Hot Start II

Hot Start Taq DNA Polymerases


Innovative design for improved preformance. Phire Hot Start II DNA Polymerase is constructed by fusing a novel DNA polymerase (orange) and a small dsDNA-binding protein (yellow). This technol-ogy dramatically increases the processivity of the polymerase thus improving its overall performance.

0 20 40 60 80 100


www.neb.com 11

Hemo KlenTaq™

Phusion® Blood Direct PCR Kit

Hemo KlenTaq™ is a truncated version of Taq DNA Polymerase that contains mutations making it resistant to inhibitors present in whole blood. Hemo KlenTaq offers the versatility and robustness of Taq and can successfully amplify samples containing up to 10% whole blood in a 25 µl reaction (20% in a 50 µl reaction).

Hemo KlenTaq™ .......................................................................................................................... M0332S/L

The Phusion® Blood Direct PCR Kit is designed for efficient amplification of DNA from various types of blood samples. This kit utilizes a modified Phusion® Hot Start High-Fidelity DNA Polymerase, a highly processive and robust PCR enzyme that is extremely tolerant of the inhibitors present in blood. This kit has been optimized for direct PCR of samples containing up to 40% whole blood from a variety of mammalian and bird species.

Phusion® Blood Direct PCR Kit ................................................................................................. F-547S/L

EXTRACTION-FREE PCR

Amplification from human whole blood with Hemo KlenTaq

Phusion Blood Direct PCR Kit delivers superior performance even at very high blood concentration

Lane 1: 5% blood + Na-EDTA; Lane 2: 5% blood + K-EDTA; Lane 3: 5% blood + Na-Heparin; Lane 4: 5% blood + Na-Citrate; Lane 5: 1 mm2 FTA Gutherie Card containing dried human blood; Lane 6: 1 mm2 FTA Card containing dried human blood (washed with 50 μl H2O). Ladder M is the 2-Log DNA Ladder (NEB #N3200).

Kits designed for direct blood PCR were compared by amplifying a 588 bp genomic DNA fragment with increasing blood concentration in the reaction mixture. PCR was performed according to suppliers’ instructions (35 cycles) using Finnzymes’ Piko® Thermal Cycler and ultra-thin walled UTW® reaction vessels. Total cycling time indicated at bottom. + and - denote control reactions.

Advantages:• Fast - No need for DNA extraction

• Robust amplification

• Dependable - Ideal for diagnostic PCR

• Versatile - Works well with most common anticoagulants, including heparin, citrate and EDTA


• Robust - Ensures accurate and specific results with high yields and short protocol times

• Dependable - Ideal for SNP analysis

• Versatile - Works well with most common anticoagulants, including heparin, citrate and EDTA

Look for the extraction-free PCR icon which indicates products from NEB that can amplify directly with no need for extraction

M 1 2 3 4 5 6kb

1.0

0.5

0.3

Blood %

1 5 10 20 30 40 - +

40 min 82 min

Phusion® Blood Direct Supplier 2

Blood %

1 5 10 20 30 40 - +

Amplification of DNA directly from starting materials such as blood or tissue has been problematic due to the presence of inhibitors in the sample. Purification of genomic DNA, as well as the use of specialized buffers or pre-treatments can overcome this difficulty, but often adds considerable time and expense to the process. As an alternative, NEB offers several kits and polymerases that can be used for direct amplification of DNA. These products offer robust activity and will save valuable laboratory time and expense.



Extraction-free PCR

12

Phire® Plant Direct PCR Kit

Phire® Animal Tissue Direct PCR Kit

The Phire Plant Direct PCR Kit is designed for the amplification of DNA directly from plant samples. This kit utilizes Phire® Hot Start II DNA Polymerase, a specially engineered enzyme with a unique DNA binding domain. Phire Hot Start II DNA Polymerase is extremely robust and tolerant of many PCR inhibitors present in plant material.

Phire® Plant Direct PCR Kit ............................................................................................................ F-130S

The Phire® Animal Tissue Direct PCR Kit has been developed for amplification of DNA directly from a wide variety of animal tissues, including mice, fish, birds and insects. This kit also utilizes Phire® Hot Start II DNA Polymerase, making it extremely robust and tolerant of many PCR inhibitors present in unpurified animal tissue.

Phire® Animal Tissue Direct PCR Kit ............................................................................................ F-140S



• Easy-to-use - Protocols require minimal sample material; sampling tools provided

• Versatile - Validated for a variety of plant species



• Easy-to-use - Protocols require minimal sample material; sampling tools provided

• Versatile - Compatible with tissues from various animal species


EXTRACTION-FREE PCR

Leaves: Lane 1: Arabidopsis, Mt DNA (1 kb); Lane 2: Arabidopsis, gDNA (3.5 kb); Lane 3: Capsicum, Mt DNA (1.4 kb); Lane 4: Maize, Mt DNA (1.4 kb). Seeds: Lanes 1-6: Mt DNA (~1.4 kb, depending on species)

Direct PCR from various animal tissues. Small samples of various animal tissues were placed directly into 50 μl PCR reactions. A specific DNA fragment was amplified with control primers included in the kit or, in the case of fruit fly and zebrafish, using control primers whose sequences are provided at the NEB website. + and – denote control reactions with or without purified DNA.

Amplification from various plant leaves and seeds with Phire Plant Direct PCR Kit

Fast and reliable PCR from various tissue samples from a number of species

M 1 2 3 4 – + +

LeavesControl DNA

M tomato maize apple – +Control DNA

Seeds

Mou

se e

ar tis

sue

M + – MBear m

uscle

tissu

e

Bird fe

athe

r

Bovine

hair

Fruit f

ly wing

Zebra

fish

fin


Visit www.neb.com/extractionfreePCR to find

more information, including application notes and

supplemental data.

www.neb.com

PCR Troubleshooting Guide

13

TROUBLESHOOTING

PROBLEM POSSIBLE CAUSE SOLUTION

SEQUENCE ERRORS

Low fidelity polymerase • Choose a higher fidelity polymerase (see selection chart on pages 2–3)

Reaction conditions are not optimal

• Reduce number of cycles• Decrease extension time• Decrease Mg++ concentration present in reaction• Increase the amount of template present

Desired sequence may be toxic to host • Clone into a non-expression vector • Use a low-copy number cloning vector

Template DNA has been damaged • Start with a fresh template

Faulty primer preparation • Repeat reaction with new primers

UV damage • UV wavelength exposure time should be limited to 254–312 nm when using a light box to analyze or excise PCR products

Unbalanced nucleotide concentrations • Prepare fresh nucleotide mix

INCORRECT PRODUCT SIZE

Mispriming • Verify that primers have no additional complementary regions within the template DNA

Improper Mg++ concentration • Adjust Mg++ concentration in 0.5 mM increments

Nuclease contamination • Repeat reactions using fresh solutions

NO PRODUCT

Primer annealing temperature too high

• If amplifying with Phusion or Phire DNA Polymerases, verify annealing temperature using the Finnzymes Tm calculator

(www.finnzymes.fi/tm_determination.html)• Lower annealing temperature in 2°C increments• Perform “Touch down” PCR (1)

Poor primer design• Check polymerase datacard/manual for recommended primer design • Verify that primers are non-complementary, both internally and to each other• Increase length of primer

Poor primer specificity • Verify that oligos are complementary to proper target sequence

Insufficient primer concentration • Increase primer concentration to 0.1–0.5 µM

Missing reaction component • Repeat reaction setup

Target sequence not present in template • Try other sources of template DNA

Poor reaction conditions

• Optimize Mg++ concentration, annealing temperature and extension time• Thoroughly mix Mg++ solution• Check primer concentrations• Perform “Touchdown” PCR

Questionable template quality • Analyze DNA via gel electrophoresis after incubation with Mg++

Presence of inhibitor in reaction

• Decrease sample volume• Purify template DNA by alcohol precipitation or drop dialysis• Try an extraction-free PCR product (see pages 11–12), designed for amplification directly in the presence of inhibitors such

as blood, plant or animal tissue

Insufficient number of cycles • Rerun the reaction with more cycles

Incorrect thermocycler programming • Check program, verify times and temperatures

Inconsistent block temperature • Test calibration of heating block

Contamination of reaction tubes or solutions

• Autoclave tubes prior to use to eliminate biological inhibitors• Prepare fresh solutions or use new reagents and new tubes

Complex template • For GC-rich templates, we recommend Phusion® DNA Polymerase with GC buffer • For longer templates, we recommend LongAmp™ Taq DNA Polymerase

MULTIPLE OR NON-SPECIFIC PRODUCTS

Premature replication (non-hot start polymerases)

• Set up reactions on ice using chilled components. Add samples to thermocycler preheated to the denaturation temperature• Try using a hot start polymerase, such as Phire Hot Start II and Phusion Hot Start II DNA Polymerases

Primer annealing temperature too low

• Raise annealing temperature in 2°C increments• If using Phusion or Phire, verify annealing temperature using Finnzymes T

m calculator

• Perform “Touchdown” PCR (1)

Insufficient mixing of reaction buffer • Thoroughly mix reaction buffer

Incorrect Mg++ concentration • Adjust Mg++ concentration in 0.5 mM increments

Poor primer design• Verify that primers are non-complementary, both internally and to each other• Increase length of primer• Avoid GC-rich 3´ ends

Excess primer • Reduce primer concentration to 0.1–0.5 µM

Contamination with exogenous DNA

• Use positive displacement pipettes or non-aerosol tips• Set-up dedicated work area and pipettor for reaction setup• Wear gloves during reaction setup

Incorrect template concentration • Use 1 pg–1 ng/50 µl rxn of phage or plasmid DNA• Use 1 ng–1 µg/50 µl rxn of genomic DNA

(1) Don, R.H. et al. (1991) Nucleic Acids Res. 19, 4008.

Visit www.neb.com to find additional tips for

optimizing PCR reactions.


cDNA Synthesis Selection Chart

DIFFICULT TEMPLATES REQUIRING HIGHER REACTION TEMPERATURE

PRODUCT NEB # FEATURES APPLICATIONS

ProtoScript® AMV First strand cDNA Synthesis Kit

E6550

• Convenient 2-tube Mix

• cDNA up to 10 kb

• Expression Analysis• Cloning• qPCR

ALL TEMPLATES

CONVENIENCE

ProtoScript® M-MuLV First strand cDNA Synthesis Kit

E6300

• Convenient 2-tube mix

• cDNA up to 13 kb


FLEXIBILITY

Individual Components:M-MuLVAMVdNTPs

M0253S/LM0277S/LN0447S/L

• AMV, M-MuLV• Selection

of Priming Strategies


14

NEB offers several reagents for cDNA sythesis for use in applications including qPCR and qRT-PCR. For your convenience, reagents are available as kits or standalone products to suit your needs.

ProtoScript® M-MuLV First Strand cDNA Synthesis Kit ................................... E6300SDesigned for the highly efficient production of cDNA copies of mRNA using M-MuLV Reverse Transcriptase (RT). ProtoScript is optimized for RT reactions using 10 pg–2 µg of total RNA.

M-MuLV Reverse Transcriptase ......... M0253S/LSynthesizes cDNA from RNA or single-stranded DNA. Isolated from a recombinant source.

ProtoScript® AMV First Strand cDNA Synthesis Kit ................................... E6550SAn optimized blend of Avian Myeloblastosis Vi rus (AMV) Reverse Transcriptase and Murine RNase Inhibitor offers a broader optimal reaction temperature.

AMV Reverse Transcriptase ........... M0277S/L/TAMV Reverse Transcriptase is an RNA-directed DNA polymerase. This en zyme can synthesize a complementary DNA strand initiating from a primer using RNA (cDNA synthesis).


cDNA SYNTHESIS

Phusion® RT-PCR Kit

Experience the benefits of Phusion® High-Fidelity DNA Polymerase in your two step RT-PCR reactions. The Phusion RT-PCR Kit enables accurate cDNA amplification with high yields and short cycling times, and is ideal for producing cDNA to be used in cloning and gene expression studies.

Phusion® RT-PCR Kit ................................................................................................................. F-546S/L

Advantages:• Robust - Generate a broad range

of RT-PCR products with high yield

• Extreme Fidelity - Amplify cDNA fragments with fidelity 50X greater than Taq

• Speed - Increased reaction speed

Phusion RT-PCR was compared to the RT-PCR systems from two major suppliers in a typical RT-PCR experiment for amplifying short cDNA fragments (192–1113 bp). Total RNA from human skeletal muscle was transcribed with random priming according to each suppliers' recommendations. Amplification of cDNA was performed with a high-fidelity DNA polymerase recommended by the suppliers. Due to the various sizes of amplicons, a PCR protocol with the lowest annealing temperature and the longest extension time defined by amplicons was used. Compared to the Phusion RT-PCR Kit, competitors systems resulted in less amplicons with varying yields. M is a molecular size marker.

Robustness of different RT-PCR Systems

bp

1353 –

603 –

194 –

M Phusion RT-PCR Kit M Supplier A M Supplier B

cDNA Synthesis

www.neb.com 15

Advantages:• Robust - Generate a broad range

of RT-PCR products with high yield

• Sensitivity - Detects transcripts as low as 20 pg total RNA

• Flexibility - Detection of multiple targets from 1 reaction

RT-PCR

Protoscript® M-MuLV Taq RT-PCR Kit

Protoscript® M-MuLV Taq RT-PCR Kit has been designed for the sensitive detection of mRNAs in a two-step process using M-MuLV Reverse Transcriptase (RT) and Taq 2X Master Mix. Protoscript M-MuLV Taq RT-PCR Kit is optimized for greater sensitivity and higher yield.

Protoscript® M-MuLV Taq RT-PCR Kit ........................................................................................ E6400S

First strand cDNA was carried out in the presence of oligo d(T)23VN, and about 1/10th of the cDNA reaction was amplified in a 35-cycle PCR reaction using the Taq 2X Master Mix (-RT control not shown).

2 µg 200 ng 20 ng 2 ng 200 pg 20 pg

RT-PCR amplification of the GAPDH gene from human spleen total RNA

1 2 3 4 5Approximately 2 μg of human spleen total RNA was reverse transcribed using d(T)23VN. Reactions were set up with and without M-MuLV Reverse Transcriptase (-RT Control). After 30 cycles of amplification using 1/20th of the cDNA product, 5 μl was analyzed on a 1% agarose gel. Lane 1: 2-log DNA Ladder, Lane 2: -RT Control of 1.2 kb fragment, Lane 3: 1.2 kb fragment approx. 15 kb from 3´ end, Lane 4: -RT Control of 0.9 kb fragment, Lane 5: 0.9 kb fragment approx. 1.1 kb from 3´ end.

Amplification of different regions of human guanine nucleotide exchange factor p532 mRNA

The ProtoScript® AMV LongAmp™ Taq RT-PCR Kit combines two powerful mixes, the AMV Enzyme Mix and the LongAmp Taq 2X Master Mix for 2-step RT-PCR applica-tions. The two mixes require minimal handling during reaction setup and yet offer consis-tent and robust RT-PCR reactions. The AMV Enzyme Mix can carry out first strand cDNA synthesis reactions with a broad optimal reaction temperature of 37°C to 50°C. LongAmp Taq 2X Master Mix offers consistent and robust amplification from more than 14 kb cDNA products. Primer d(T)

23VN is included for anchored priming at the polyA-tail region

of messenger RNAs. Random Primer Mix is an optimized mix of hexamer and d(T)23

VN primers, offering complete coverage of RNA templates.

Protoscript® AMV LongAmp™ Taq RT-PCR Kit ......................................................................... E5300S

Protoscript® AMV LongAmp™ Taq RT-PCR Kits

Advantages:• Robust - Semi-quantitative expression

analysis of messenger RNAs by agarose gel electrophoresis

• High Yield - Suitable for a broad range of RT-PCR products

• Convenience - Two mixes offer optimal results with simple reaction setup


16

PreCR™ Repair MixThe PreCR™ Repair Mix is a cocktail of enzymes formulated to repair damaged DNA in vitro prior to PCR. The repair pre-treatment can be applied to techniques such as whole genome amplification, DNA sequencing and microarray analysis.

The PreCR Repair Mix uses enzymes in a coordinated process that emulates base excision repair. The heart of the mix is a combination of polymerase and ligase activities. The ligase acts strongly on nicks but has no detectable activity on blunt ends to minimize chi-meric gene formation. Added to this core are enzymes that recognize different DNA lesions.

The PreCR Repair Mix can repair a wide range of damaged DNA, resulting from exposure to heat, low pH, oxygen, and/or UV light. The lesions repaired by the PreCR Repair Mix do not include all possible damages. Currently, it cannot repair DNA crosslinks, such as those that occur during exposure to formalin, nor can the mix effectively repair highly fragmented DNA.

PreCR™ Repair Mix ................................................................................................................... M0309S/L

Advantages:• Flexible - Suitable for PCR,

microarrays and other DNA technologies

• Specific - Treats damaged DNA without harming template

• Versatile - Can be used in conjunction with any thermophilic polymerase

• Direct - PCR can be done directly on repair reaction

PreCR can be used to repair:• Abasic sites

• Nicks

• Thymidine dimers

• Blocked 3´ ends

• Oxidized guanine

• Oxidized pyrimidines

• Deaminated cytosine

COMPANION PRODUCTS

Proven repair with the PreCR Repair Mix The gel shows trial amplifications from damaged DNA that was either not treated (-) or treated (+) with the PreCR Repair Mix. Type of DNA damage is shown. Note: heat treated DNA is incubated at 99°C for 3 minutes. Marker M is the 2-Log DNA Ladder (NEB #N3200).

kb

10 –

3.0 –

1.0 –

0.5 –

PreCR Treatment – + – + – + – + – + – +

M A B C D E F

M: 2-Log DNA Ladder (NEB #N3200)

A: UV exposure (l DNA)

B: heat treatment (l DNA)

C: oxidation (plasmid)

D: UV exposure (human genomic DNA)

E: hydrolysis & UV exposure (l DNA)

F: hydrolysis (l DNA)

Visit confidentPCR.com to find the ideal polymerase for your applicationThere are already enough variables in PCR, don’t let polymerase performance be one of them. Choose from one of the largest selections of polymerases for PCR applications from the leader in enzyme technology and bring unparalleled confidence to your experiments.


NEB recommends the highlighted polymerase for this application. Depending on particular parameters, experiments may benefi t from one of the alternative enzymes presented. Use the provided characteristics to choose the ideal enzyme for your experiment.

Recommended Polymerases for Routine PCR:

Routine PCR

HighFidelity

High Yield

Hot Start

FastLong

Amplicon

Difficult (GC-rich) Templates

High Throughput

Multiplex PCR

Extraction-free PCR

DNA-labeling

Site-directed

Mutagenesis

Phusion™

DNA Polymerase

Phusion™

Hot StartII DNA

Polymerase

Phusion™

FlashDNA

Polymerase

Deep VentR®

DNAPolymerase

VentR®

DNAPolymerase

LongAmpTaq DNA

Polymerase

Phire HotStart II

DNAPolymerase

Taq DNAPolymerase

Crimson Taq DNA

Polymerase

DyNAzyme II Hot Start

DNAPolymerase

Fidelity vs. Taq 50X 50X 25X 6X 5X 2X 2X 1X 1X 1X

Amplicon Size ≤20 kb ≤20 kb ≤20 kb ≤6 kb ≤6 kb ≤40 kb ≤7.5 kb ≤5 kb ≤5 kb ≤3 kb

Kit Available Yes No No No No Yes No Yes No No

Master Mix Available Yes No Yes No No Yes No Yes No No

Advantages

Extreme fidelity &

robustness, use for GC-rich

templates

hot start for

improved specificity

extreme speed &

fidelity, use for GC-rich templates

ideal for GR-rich

or looped sequences

thermostability & fidelity

more robust

& longer amplicons than Taq, use for GC-rich

templates

fast hot start with high yield

industry standard for routine PCR

direct gel loading & tracking

room T reaction setup &

increased specificity

www.neb.com 17

Advantages:• Stable at room temperature

• Sharp uniform bands with easy-to- identify reference bands

• Can be used for sample quantitation

• Exceptional value

COMPANION PRODUCTS

DNA Ladders from New England Biolabs

DNA LaddersChoose DNA ladders from New England Biolabs and take the guesswork out of your DNA sample analysis. Select the ladder to suit your experimental needs and let our sharp, evenly-spaced bands help you achieve accuracy in your analysis. For your convenience, supplemental loading dye is provided with all ladders. In addition, our most popular ladders are available in a Quick-Load™ (contains blue dye to track migration) and a TriDye™ (contains three dyes) format.

Nucleotide SolutionsDeoxynucleotide Solution Set ................. N0446SThe Deoxynucleotide Solution Set contains four separate 100 mM solutions of ultrapure nucleotides (dATP, dCTP, dGTP, and dTTP).

Deoxynucleotide Solution Mix .......... N0447S/LThe Deoxynucleotide Solution Mix is an equimolar mixture of ultrapure dATP, dCTP, dGTP, and dTTP. Each nucleotide is present at a concentration of 10 mM in the mixture for a total dNTP concentration of 40 mM.

7-deaza-dGTP* .......................................... N0445SA useful additive for PCR of GC-rich templates; contains a 5 mM solution of 7-deaza-dGPT as a dilithium salt.* licensed from Roche Diagnostics GmbH

Acyclonucleotide Set ................................ N0460SAcyclonucleotide Set contains four separate tubes of acyNTPs (acyATP, acyCTP, acyGTP and acyTTP).

* Available in Quick-Load and Tri-Dye formats ** Available in Quick-Load format

1 kb DNA Ladder*

0.8% TAE agarose gel.

NEB #N3232

kb10.08.0

6.05.0

4.0

3.0

2.0

1.5

1.0

0.5

100 bp DNA Ladder*

1.3% TAE agarose gel.

NEB #N3231

bp1,517

1,200

1,000

900

800

700

600

500/517

400

300

200

100

2-Log DNA Ladder*

1.0% TBE agarose gel.

NEB #N3200

kb10.08.06.05.04.03.0

2.0

1.5

1.2

1.00.90.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Low Molecular Weight DNA Ladder**

1.8% TBE agarose gel. NEB #N3233

bp766

500

350

300

250

200

150

100

75

50

25

50 bp DNA Ladder**


NEB #N3236

bp1,350

916

766700650600550500450

400

350

300

250

200

150

100

50

Fast DNA Ladder


NEB #N3238

kb10.05.0

3.0

2.0

1.5

1.0

.766

.500

.300

.150

0.050

Mass DNA Ladder

1% TBE agarose gel.

NEB #N3237

kb10.0

6.0

4.0

3.0

2.0

1.0

0.5

PCR Marker


NEB #N3234

bp766

500

300

150

50


18

ORDERING INFORMATION

PRODUCT NEB # SIZE

Crimson LongAmp™ Taq DNA Polymerase M0326S/L 250/1,250 units

Crimson Taq DNA Polymerase M0324S/L 200/1,000 units

Crimson Taq DNA Polymerase with (Mg-free) Buffer M0325S/L 200/1,000 units

Deep VentR

™ DNA Polymerase M0258S/L 200/1,000 units

Deep VentR

™ (exo-) DNA Polymerase M0259S/L 200/1,000 units

DyNAzyme™ EXT DNA Polymerase F-505S/L 200/1,000 units

DyNAzyme™ EXT PCR Kit F-552S/L 200 rxns/1,000 rxns (50 µl rxn vol)

DyNAzyme™ II Hot Start DNA Polymerase F-504S/L 250/1,000 units

Hemo KlenTaq™ DNA Polymerase M0332S/L 200/1,000 rxns (25 µl rxn vol)

LongAmp™ Taq 2X Master Mix M0287S/L 100 rxns/500 rxns (50 µl rxn vol)

LongAmp™ Taq DNA Polymerase M0323S/L 500/2,500 units

LongAmp™ Taq PCR Kit E5200S 100 rxns (50 µl rxn vol)

Multiplex PCR 5X Master Mix M0284S 100 rxns (50 µl rxn vol)

Phire® Animal Tissue Direct PCR Kit F-140S 200 rxns (50 µl rxn vol)

Phire® Hot Start II DNA Polymerase F-122S/L 200/1,000 rxns (50 µl rxn vol)

Phire® Plant Direct PCR Kit F-130S/L 200 rxns (50 µl rxn vol)

Phusion® Blood Direct PCR Kit F-547S/L 100/500 rxns (20 µl rxn vol)

Phusion® Flash High-Fidelity PCR Master Mix F-548S/L 100 rxns/500 rxns (20 µl rxn vol)

Phusion® High-Fidelity DNA Polymerase F-530S/L 100/500 units

Phusion® High-Fidelity PCR Master Mix with HF Buffer F-531S/L 100 rxns/500 rxns (50 µl rxn vol)

Phusion® High-Fidelity PCR Master Mix with GC Buffer F-532S/L 100 rxns/500 rxns (50 µl rxn vol)

Phusion® High-Fidelity PCR Kit F-553S/L 50 rxns/200 rxns (50 µl rxn vol)

Phusion® Hot Start II High-Fidelity DNA Polymerase F-549S/L 100/500 units

Phusion® Site-Directed Mutagenesis Kit F-541S 20 rxns

QuickLoad™ Taq 2X Master Mix M0271S/L 100 rxns/500 rxns (50 µl rxn vol)

Taq 2X Master Mix M0270S/L 100 rxns/500 rxns (50 µl rxn vol)

Taq 5X Master Mix M0285S/L 100/500 reactions (50 µl rxn vol)

Taq DNA Polymerase with Standard Taq Buffer M0273S/L/X 400/2,000/4,000 units

Taq DNA Polymerase with Standard Taq (Mg-free) Buffer M0320S/L 400/2,000 units

Taq DNA Polymerase with ThermoPol Buffer M0267S/L/X 400/2,000/4,000 units

Taq DNA Polymerase with ThermoPol II (Mg-free) Buffer M0321S/L 400/2,000 units

Taq PCR Kit E5000S 200 rxns (100 µl rxn vol)

VentR

® DNA Polymerase M0254S/L 200/1,000 units

VentR

® (exo-) DNA Polymerase M0257S/L 200/1,000 units

PCR Polymerases

www.neb.com 19

ORDERING INFORMATION

PreCR™ Repair Mix M0309S/L 30/150 rxns

DNA Repair

1 kb DNA Ladder N3232S/L 200/1,000 gel lanes

100 bp DNA Ladder N3231S/L 100/500 gel lanes

2-Log DNA Ladder (0.1–10.0 kb) N3200S/L 100/500 gel lanes

50 bp DNA Ladder N3236S/L 100/500 gel lanes

Low Molecular Weight DNA Ladder N3233S/L 100/500 gel lanes

Fast DNA Ladder N3238S 100/500 gel lanes

Mass DNA Ladder N3237S 50 gel lanes

PCR Marker N3234S 50 gel lanes

Deoxynucleotide Solution Set N0446S 25 µmol of each

Deoxynucleotide Solution Mix N0447S/L 8 µmol of each/40 µmol of each

Acyclonucleotide Set N0460S 0.5 µmol of each

7-deaza-dGTP N0445S 0.15 µmol

M-MuLV Reverse Transcriptase M0253S/L 10,000/50,000 units

AMV Reverse Transcriptase M0277S/L/T200/1,000/500 units S/L (10,000 units/ml) – T (25,000 units/ml)

DyNAmo™ cDNA Synthesis Kit F-470S/L 20/100 rxns

Companion Products

PRODUCT NEB # SIZE

Phusion® RT-PCR Kit F-546S/L 20/100 rxns

ProtoScript® AMV LongAmp™ Taq RT-PCR Kit E5300S 30 rxns

ProtoScript® M-MuLV Taq RT-PCR Kit E6400S 30 rxns

ProtoScript® M-MuLV First Strand cDNA Synthesis Kit E6300S 30 rxns

ProtoScript® AMV First Strand cDNA Synthesis Kit E6550S 30 rxns

RT-PCR & qRT-PCR

For licensing information, visit www.neb.com.

USA

New England Biolabs, Inc.

Telephone: (978) 927-5054

Toll Free: (U.S. Orders) 1-800-632-5227

Toll Free: (U.S. Tech) 1-800-632-7799

Fax: (978) 921-1350

[email protected]

Canada

New England Biolabs, Ltd.

Toll Free: 1-800-387-1095

[email protected]

China, People’s Republic

New England Biolabs (Beijing), Ltd.

Telephone: 010-82378265/82378266

[email protected]

Germany

New England Biolabs GmbH

Free Call: 0800/246 5227

[email protected]

United Kingdom

New England Biolabs (UK), Ltd.

Call Free 0800 318486

[email protected]

Japan

New England Biolabs Japan, Inc.

Telephone: +81 (0)3 5669 6191

[email protected]

www.neb.com

New England Biolabs, Inc.

240 County Road

Ipswich, MA 01938-2723

Version 2.0PCR

Documents

PCR REAGENTS PCR & RT-PCRbiology.hunter.cuny.edu/molecularbio/Class... · (2) Use Multiplex PCR 5X Master Mix. (3) ™Use Quick-Load Taq 2X Master Mix. (4) ™Use Crimson LongAmp