Now includes

NEB® Golden Gate Assembly Kit (BsaI-HF®v2)

Reagents and Tools for Molecular Cloning

CLONE WITH CONFIDENCE ®

be INSPIRED
drive DISCOVERY
stay GENUINE
 REAGENTS & TOOLS FOR MOLECULAR CLONING

Clone with confidence ®

Built on over 40 years of experience, New England Biolabs® provides you with the quality reagents and technical support
that you need to take your cloning experiments to the next level. With reagents for each step in the cloning workflow,
NEB® products deliver quality and performance, so that you can clone with confidence.

Cloning INSERT PREPARATION

Workflow Starting
Material
RNA
Comparison OR OR OR OR cDNA Synthesis

Plasmid gDNA PCR products Annealed oligos cDNA

Traditional Cloning OR PCR Cloning OR Seamless Cloning OR LIC (Ligation

(RE Digestion & Ligation) (TA & Blunt-End) (Gene Assembly) Independent Cloning)

RE Digestion PCR PCR PCR

DNA
Preparation 60 min. (Standard) 90 min. 90 min. 90 min.
5–15 min. (Time-Saver)

dsDNA P
intermediate P OR OR OR OR
A A A
A A A
P
Dephosphorylation Clean Up Clean Up Clean Up
Blunting 15 min. 15 min. 15 min.
(Optional)
DNA End Phosphorylation Cohesive-End Cohesive-End
10–30 min.
Modifications (Optional) Formation by Formation by
30 min. 5´ → 3´ exo 3´ → 5´ exo
P 30 min. 30 min.

dsDNA P
P OR P OR OR
intermediate 2 P
A
A
P P P
Gel and Column
Purification
Vector & Insert 75 min. Ligation Ligation Annealing
Joining
15 min. Occurs simultaneously 30 min.
Ligation with previous step
Instant – 15 min.

T A
A T

OR OR

Assembled vector

Estimated total time* 1 hr., 20 min. – 3 hr. 2 hr. – 2 hr., 30 min. 2 hr., 15 min. 2 hr., 45 min.

* Note that times are based on estimates for moving a gene from one plasmid to another.
If the source for gene transfer is gDNA, add 2 hours to calculation for the traditional
cloning method. Total time does not include transformation, isolation or analysis. Transformation

** 70 minutes for recombination occurs on second day

2
12
 REAGENTS & TOOLS FOR MOLECULAR CLONING

The figure below compares the various cloning methodologies. To help get you started with your experiment, we have
included selection charts for each step in the workflows, as well as recommended products and tips to ensure successful
cloning experiments. To learn more about the full portfolio of products available, visit CloneWithNEB.com.

VECTOR PREPARATION

Multiple cloning site

Starting (MCS)
Material

Plasmid

OR Recombinational Restriction Enzyme OR

PCR
(Gateway/Creator/Univector) (RE) Digestion

PCR RE Digestion PCR

DNA
90 min. Processing 60 min. (Standard) 2 hr.
5–15 min. (Time-Saver)
Clean Up
15 min.
P P

P P
Clean Up OR
15 min.

Linear vector

Dephosphorylation (Optional) T-addition

RE Digest DNA End
10–30 min. 1.5 hr.
60 min. (Standard) Modifications
5–15 min. (Time-Saver)
Clean Up 15 min OR
Gel & Column Purification 75 min.

+
T
T

Clean Up OR
15 min.
Site-Specific
Recombination Linear vector,
60 min. ready for joining

Proteinase K Treatment
Recombination 10 min. Estimated total time 20 min. – 2 hr., 25 min. 3 hr., 45 min.
sites

70 min.**

Holding vector
Endpoint vector

3 hr., 15 min. – 5 hr., 20 min.

DNA Isolation DNA

(Plasmid Purification) Analysis Protein
Expression

Functional
OR OR
Analysis

RE Digest Colony PCR Sequencing Site-Directed

Mutagenesis
3
 REAGENTS & TOOLS FOR MOLECULAR CLONING

NEBuilder HiFi DNA Assembly ®

NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA RECOMMENDED PRODUCTS
fragments, even those with 5´- and 3´-end mismatches. Available with and
without competent E. coli, this flexible kit enables simple and fast seamless NEBuilder HiFi DNA Assembly Cloning Kit
cloning utilizing a proprietary high-fidelity polymerase. Make NEBuilder HiFi (NEB #E5520)
your first choice for DNA assembly and cloning. NEBuilder HiFi DNA Assembly Master Mix
(NEB #E2621)
Overview of the NEBuilder HiFi DNA Assembly cloning method NEBuilder HiFi DNA Assembly Bundle for
Large Fragments (NEB #E2623)
DNA Preparation • Simple and fast seamless cloning in as little as 15 minutes
C
From:
B • U
 se one system for both "standard-size" cloning and larger
+
• PCR Linear A
• Restriction enzyme vector gene assembly products (up to 11 fragments and 20 kb)
digestion DNA inserts with 15-30 bp
• Synthetic DNA overlapping ends (PCR-amplified) • D
 NA can be used immediately for transformation
(e.g., gBlocks)
• Single-stranded oligo or as template for PCR or RCA
• A dapts easily for multiple DNA manipulations,
NEBuilder HiFi DNA B
Assembly Master Mix A C
including site-directed mutagenesis
Single-tube reaction • E njoy less screening/re-sequencing of constructs,
• Exonuclease chews Assembled
back 5´ ends to create DNA with virtually error-free, high-fidelity assembly
single-stranded 3´ overhangs
• DNA polymerase fills in gaps within Incubate at 50°C
• U
 se NEBuilder HiFi in successive rounds of assembly,
each annealed fragment for 15-60 minutes
as it removes 5´- and 3´-end mismatches
• DNA ligase seals nicks
in the assembled DNA
• B ridge two ds-fragments with a synthetic ssDNA oligo
for simple and fast construction (e.g., linker insertion
Transformation
or gRNA library)
• No
 licensing fee requirements from NEB for
NEBuilder products
DNA Analysis • NEBuilder
 HiFi DNA Assembly Cloning Kit includes
OR OR
the NEBuilder HiFi DNA Assembly Master Mix and
RE Digest Colony PCR Sequencing
NEB 5-alpha Competent E. coli
• NEBuilder
 HiFi DNA Assembly Bundle for Large Fragments
includes the NEBuilder HiFi DNA Assembly Master Mix
NEBuilder HiFi DNA Assembly
and NEB 10-beta Competent E. coli for assemblies larger
can be used for a variety of DNA assembly methods.
than 15 kb.

2-fragment assembly ssOligo & dsDNA assembly

TOOLS & RESOURCES

Visit NEBuilderHiFi.com to find:

• Online tutorials to help with assembly and primer design

4-fragment assembly Annealed-oligo assembly • Application notes utilizing NEBuilder HiFi

15-bp overlap 25-bp overlap • A ccess to NEBuilder Assembly Tool, our online primer
Sticky end Blunt end
design tool at NEBuilder.neb.com

High efficiency
DOWNLOAD THE NEB AR APP*

3´- and 5´-end mismatch assembly Site-directed mutagenesis

Multiple sites
How does
NEBuilder HiFi DNA
Assembly work?

*see back cover for details

4
 REAGENTS & TOOLS FOR MOLECULAR CLONING

Golden Gate Assembly

The efficient and seamless assembly of DNA fragments, commonly referred to as RECOMMENDED PRODUCTS
Golden Gate assembly (1,2), has its origins in 1996 when, for the first time, it
was shown that multiple inserts could be assembled into a vector backbone using NEB Golden Gate Assembly Kit (BsaI-HF®v2) (NEB #E1601)
only the sequential (3) or simultaneous (4) activities of a single type IIS restriction • U
 pdated to include BsaI-HFv2 (optimized for Golden Gate)
enzyme and T4 DNA Ligase. Since this pioneering work, Golden Gate has • S eamless cloning – no scar remains following assembly
enabled single inserts, the cloning of inserts from diverse populations enabling
• Includes destination plasmid with T7/SP6 promoters
library creation, and multi-module assemblies. We now have made extraordinary
• Ordered
 assembly of multiple fragments (2-20+)
improvements that touch every application of the Golden Gate technology.
in a single reaction
• C
 an also be used for cloning of single inserts and library
Advances in Ligase Fidelity preparations
Research at NEB has led to increased understanding of ligase fidelity, including • Efficient
 with regions with high GC content and
the development of a comprehensive method for profiling end-joining ligation areas of repeats
fidelity in order to predict which overhangs have improved fidelity (5). This • C
 ompatible with a broad range of fragment sizes
research allows careful choice of overhang sets, which is especially important for (< 100 bp to > 15 kb)
achieving complex Golden Gate Assemblies.
Type IIS Enzymes used in Golden Gate
- BsaI (NEB #R0535)
Type IIS Restriction Enzymes for Golden Gate Assembly - BsaI-HFv2 (NEB #R3733)
NEB offers more Type IIS (i.e., recognize asymmetric DNA sequences and cleave - BbsI (NEB #R0539)
outside of their recognition sequence) restriction enzymes than any other supplier, - BbsI-HF (NEB #R3539)
- BsmBI (NEB #R0580)
many of which are used in Golden Gate Assembly. NEB is pleased to introduce
- Esp3I (NEB #R0734)
two new restriction enzymes for use in Golden Gate: Esp3I, an isoschizomer of
BsmBI that is recommended for use at 37°C and is supplied with CutSmart® TOOLS & RESOURCES
Buffer, and the improved BsaI-HFv2, optimized for Golden Gate Assembly.
This enzyme along with the ligase fidelity data, allows complex 20+ fragment Visit www.neb.com/GoldenGate to find:
assemblies with high efficiency, > 90% accuracy and low backgrounds. • Publications
 and protocols related to ligase fidelity and
Golden Gate Assembly
Golden Gate Assembly Workflow for complex assemblies • A ccess to NEB Golden Gate Assembly Tool,
for help with designing your experiment at
BsaI-HFv2
P1
GoldenGate.neb.com
5´ GGTCTCNNNNN 3´

CCAGAGNNNNN 5´ GGTCTCNNNNN NNNNNGAGACC 3´

• Access
 to the Ligase Fidelity Viewer to visualize
3´ 5´ Insert
3´ CCAGAGNNNNN NNNNNCTCTGG 5´ fragment A

GGT P2 overhang ligation preferences for T4 DNA Ligase under

ACC
A G GG
N G TCT
CCA C T C
GA N
GN N
+ P3
experimental conditions
N N NNC N
N 5´ GGTCTCNNNNN NNNNNGAGACC 3´
N N NN

Insert
NN N

N
N

Destination CCAGAGNNNNN NNNNNCTCTGG 5´ fragment B

• View
 our webinar: Fidelity and bias in end-joining ligation:
3´
vector
P4
Enabling complex multi-fragment Golden Gate DNA
PCR amplification of fragments
Assembly at www.neb.com/NEBTVwebinars

A B References:
Single-tube
BsaI-HFv2- + + Assembled 1. Engler, C. et al. (2008) PLoS ONE, 3: e3647.
reaction
digested vector
PCR-amplified fragments,
DNA product 2. Engler, C. et al. (2009) PLoS ONE, 4: e5553.
• BsaI-HFv2 BsaI-HFv2-digested 3. Lee, J.H. et al. (1996) Genetic Analysis: Biomolecular Engineering, 13; 139-145.
• DNA ligase
4. Padgett, K.A. and Sorge, J.A. (1996) Gene, 168, 31-35.
Can be used in complex
(20+) fragment assemblies 5. Potapov, V. et. al. (2018) ACS Synth. Biol. DOI: 10.1021/acssynbio.8b00333.

In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, BsaI-HFv2 (GGTCTC),
added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing
the designed 4-base overhangs that direct the assembly. How does
Golden Gate
Assembly work?

Gibson Assembly ®

RECOMMENDED PRODUCTS
Gibson Assembly enables multiple, overlapping DNA fragments to be joined in a
single-tube isothermal reaction, with no additional sequence added (scar-less). Gibson Assembly Cloning Kit (NEB #E5510)
Gibson Assembly Master Mix (NEB #E2611)
Visit NEBGibson.com for more information.
5
 REAGENTS & TOOLS FOR MOLECULAR CLONING

Comparison of DNA Assembly Reaction Types

* Assembled products are treated with T5 exonuclease followed by PCR. Only covalently
NEBuilder HiFi NEB Gibson sealed products resistant to T5 exonuclease digestion can serve as templates for PCR
DNA Assembly Assembly In-Fusion® HD and yield PCR product.
+++ Performs best; recommended X Does not perform
Assembly Covalently Assembly Covalently Assembly Covalently
++ Performs well; but other product(s) perform better NP Experiment not performed
ASSEMBLY REACTION TYPES efficiency sealed?* efficiency sealed?* efficiency sealed?*
+ Performs, but not recommended
2-fragment assembly

No mismatch +++ Yes ++ Yes ++ No

3´- and 5´-end mismatch +++ Yes ++ Yes X No
4-fragment assembly

15-bp overlap & no mismatch +++ Yes ++ Yes ++ No

25-bp overlap & no mismatch +++ Yes ++ Yes ++ No
Oligo assembly NEB offers several online tools
3´- and 5´-overhang +++ Yes ++ Yes X No to aid in DNA assembly. Visit
Blunt end & no mismatch +++ Yes ++ Yes X No the Tools & Resources tab at
ssOligo & vector +++ Yes NP Yes X No www.neb.com for the full list.

DNA Assembly Selection Chart

NEBuilder HiFi Gibson NEB Golden Gate USER® Enzyme
DNA Assembly Assembly Assembly Kit
(NEB #M5505)
(BsaI-HFv2)
(NEB #E2621) (NEB #E5510)
Thermolabile
(NEB #E5520) (NEB #E2611) (NEB #E1601)
USER II Enzyme
(NEB #E2623)
(NEB #M5508)
PROPERTIES

Removes 5´ or 3´ End Mismatches

*** * N/A N/A

Assembles with High Fidelity at Junctions

*** ** *** ***
Tolerates Repetitive Sequences at Ends
* * *** ***
Generates Fully Ligated Product
*** *** *** NR

Joins dsDNA with Single-stranded Oligo

*** ** NR NR

Assembles with High Efficiency with Low Amounts of DNA

*** ** ** **
Accommodates Flexible Overlap Lengths
*** *** * **
APPLICATIONS

Simple Cloning (1-2 Fragments)

*** *** *** ***
4-6 Fragment Assembly (one pot)
*** *** *** ***
7-11 Fragment Assembly (one pot)
*** ** *** ***
12-24 Fragment Assembly (one pot)(1) * * *** NR

Template Construction for In vitro Transcription

*** *** *** ***
Synthetic Whole Genome Assembly
*** * * *
Multiple Site-directed Mutagenesis
*** ** ** **
KEY
Library Generation
*** *** *** **
Optimal, recommended product for
Metabolic Pathway Engineering
*** ** *** *** *** selected application
TALENs
** ** *** ** ** Works well for selected application

Short Hairpin RNA Cloning (shRNA)

*** ** * * * Will perform selected application,
but is not recommended
gRNA Library Generation
*** ** * * (1) Please visit neb.com/GoldenGate for
Large Fragment (> 10 kb) Assembly
*** *** *** ** more information

N/A Not applicable to this application

Small Fragment (< 100 bp) Assembly
*** * *** *** NR Not recommended

6
Use in Successive Rounds of Restriction Enzyme Assembly
*** * NR
*
 REAGENTS & TOOLS FOR MOLECULAR CLONING

NEB PCR Cloning Kit ®

The NEB PCR Cloning Kit enables quick and simple cloning of all your PCR RECOMMENDED PRODUCTS
amplicons, regardless of the polymerase used. This kit utilizes a novel mechanism
for background colony suppression – a toxic minigene is generated when the NEB PCR Cloning Kit (NEB #E1202)
vector closes upon itself – and allows for direct cloning from your reaction, with NEB PCR Cloning Kit
no purification step. (without competent cells) (NEB #E1203)

Cloning Kit Protocol Overview • Easy cloning of all PCR products, including blunt and
TA ends
Mix insert with 1 µl linearized
vector; bring to 5 µl with H2O • Fast cloning experiments with 5-minute ligation step

• Simplified screening with low/no colony background

Ligation

and no blue/white selection required

Add 4 µl Cloning Mix 1
and 1 µl Cloning Mix 2

Incubate at room
• Save time by eliminating purification steps
temperature (25°C)
for 5-15 minutes* • Updated to allow for in vitro transcription with both
Incubate on ice for 2 minutes
SP6 and T7 promoters
Add 2 µl of completed ligation
Transformation

reaction to 50 µl competent cells • Flanking restriction sites available for easy subcloning,
Incubate on ice including choice of two single digest options
for 20 minutes

Heat shock at 42°C for 30 seconds • Provided analysis primers allow for downstream colony
Incubate on ice Applies to
Competent Cells
PCR screening or sequencing
for 5 minutes
Supplied with
Add 950 µl NEB 10-beta/Stable
Outgrowth Medium for outgrowth
the NEB PCR • Ready-to-use kit components include 1 kb control
Cloning Kit
Incubate at 37°C (NEB #E1202) amplicon, linearized cloning vector and optional
for 1 hour
single-use competent E. coli
Plating

Spread 50 µl and 50 µl of a 1:10 dilution on

ampicillin selection plate; incubate at 37°C overnight
• Longer shelf life (12 months), as compared to some
*Note: While 5 minutes is recommended, 15 minutes
will increase transformation levels for inserts suspected
as being difficult to clone.
commercially available products

Vector Maps and Sequence • Value pricing

BtgZI 100
SapI 2526 BsaXI 2541 AfeI 144
PciI - AflIII 2409 NdeI 208
NruI 226
DrdI 2307 SbfI - PstI 464
BsaJI 2249 PmeI 473
Constitutive PspXI - XhoI 510
Promoter BamHI 516
Sites EcoRI 522
BseYI 2105 PacI 561
Flanking ZraI 567
Toxic Minigene AatII 569
ori

AlwNI 2000 Cloning Site EcoRI 572

XhoI 577
pMiniT™ 2.0 NotI- EagI 584
2,588 bp BsmBI 602

SspI 716

R
Ap XmnI 921
BanI 1568
AhdI 1521 BcgI 1017
BmrI 1481
FspI 1298 ScaI - RsaI 1040
BpmI 1452
BsrFI 1436 PvuI 1152
BglI 1403
NmeAIII 1373

Features within Sequence Flanking the Toxic Minigene/Cloning Site

Cloning Analysis Forward Primer
SbfI/PstI PmeI
5´ ACC TGC CAA CCA AAG CGA GAA CAA AAC ATA ACA TCA AAC GAA TCG ACC GAT TGT TAG GTA ATC GTC ACC TGC AGG AAG GTT
3´ TGG ACG GTT GGT TTC GCT CTT GTT TTG TAT TGT AGT TTG CTT AGC TGG CTA ACA ATC CAT TAG CAG TGG ACG TCC TTC CAA

+1
SP6 Promoter
PmeI PspXI/ XhoI BamHI EcoRI
5´ TAA ACG CAT TTA GGT GAC ACT ATA GAA GTG TGT ATC GCT CGA GGG ATC CGA ATT CAG GAG GTA AAA ACC
3´ ATT TGC GTA AAT CCA CTG TGA TAT CTT CAC ACA TAG CGA GCT CCC TAG GCT TAA GTC CTC CAT TTT TGG

...ATG AT C TGA TAA TAA...

...TAC TA INSERT G ACT ATT ATT...

Met Ile (interrupted) * * *

Two Amino Acid Toxic Minigene with Cloning Site Shown

(As diagrammed, minigene inactivated if insert cloned into site)

Map shown displays the construct formed if no insert

PacI ZraI/AatII EcoRI/XhoI NotI/EagI BsmBI
is present. Unique restriction sites are shown in
5´ TTA ATT AAG ACG TCA GAA TTC TCG AGG CGG CCG CAT GTG CGT CTC CCT ATA GTG AGT CGT ATT AAT TTC GCG GGC
3´ AAT TAA TTC TGC AGT CTT AAG AGC TCC GCC GGC GTA CAC GCA GAG GGA TAT CAC TCA GCA TAA TTA AAG CGC CCG bold. Additional restriction sites that can be used for
+1 subcloning are also shown. Expanded box below shows How does
T7 Promoter location of sequencing primers, restriction sites for the NEB PCR
5´ GGA ACC CCT ATT TGT TTA TTT TTC TAA ATA CAT TCA AAT ATG TAT CCG CTC ATG AGA CAA TAA CCC TGA 3´
3´ CCT TGG GGA TAA ACA AAT AAA AAG ATT TAT GTA AGT TTA TAC ATA GGC GAG TAC TCT GTT ATT GGG ACT 5´ subcloning or linearization for in vitro transcription, Cloning Kit work?
Cloning Analysis Reverse Primer
RNA Polymerase promoter sequences and placement of
insertion site within the toxic minigene.
7
 REAGENTS & TOOLS FOR MOLECULAR CLONING

Q5 Site-Directed Mutagenesis Kit

®

The Q5 Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of RECOMMENDED PRODUCTS
double-stranded plasmid DNA in less than 2 hours. The kit utilizes Q5 Hot Start
High-Fidelity DNA Polymerase, along with custom mutagenic primers to create Q5 Site-Directed Mutagenesis Kit (NEB #E0554)
substitutions, deletions and insertions in your vector of choice. Transformation Q5 Site-Directed Mutagenesis Kit
into high-efficiency NEB 5-alpha Competent E. coli cells ensures robust results (Without Competent Cells) (NEB #E0552)
with plasmids up to 14.3 kb in length. KLD Enzyme Mix (NEB #M0554)
Overview of Q5 Site-Directed Mutagenesis Kit • G
 eneration of mutations, insertions or deletions
in plasmid DNA

*
1. Exponential Amplification
(PCR)
2. Treatment and Enrichment:
Kinase, Ligase and DpnI
3. Transformation • Non-overlapping primer design ensures robust, exponential
• NEB 5-alpha
• Q5 Hot Start • 10X KLD Competent Cells amplification and generates a high % of desired mutations
2X Master Mix Enzyme Mix (included in NEB #E0554)

Substitutions • Low error rate of Q5 High-Fidelity DNA Polymerase reduces

screening time
• Use of standard primers eliminates need for phosphorylated
Deletions
or purified oligos

5 min. at room temp.

• Easy-to-use master mix format
2A. Phosphorylation 2B. Ligation
*
2C. Template Removal • For help with primer design, try NEBaseChanger™
at NEBaseChanger.neb.com
* *
*
**

P P *
**

*
*

Insertions
*
**

P P
**

*
*
*

*
**

P P
**

Learn more about

the benefits of
the Q5 SDM Kit.

Introducing: Luna® Universal

qPCR & RT-qPCR Products
Rapid, sensitive and precise dye- or probe-based detection
and quantitation of DNA and RNA
• Compatible across a wide variety of instrument platforms
• Visible tracking dye reduces pipetting errors
• Novel thermostable reverse transcriptase
improves performance
• One-step RT-qPCR reduces workflow time be INSPIRED
drive DISCOVERY
Visit LUNAqPCR.com to learn more. stay GENUINE

8
 PCR

Amplify your confidence with our PCR polymerases

Whether you are a seasoned cloner or a first-timer, choosing the optimal PCR RECOMMENDED PRODUCTS
polymerase for your target amplicon is essential. With NEB’s selection of poly-
merases for standard, high-fidelity and specialty PCR, you can have confidence Q5 High-Fidelity DNA Polymerase
in the reliability of amplification, regardless of sequence composition. For exam-
• ~280X the fidelity of Taq DNA Polymerase
ple, our high-fidelity polymerases ensure accuracy in the amplification of your
insert. However, when fidelity is less important, such as for screening or colony • H
 igh specificity and robust yields with minimal
optimization
PCR, you’ll get robust yields with minimal optimization using one of our routine
polymerases, all at a lower price. Add flexibility to your reaction setup with • S uperior performance for a broad range of amplicons
enzyme, kit or master mix formats. Let NEB’s PCR Polymerases amplify your (from high AT to high GC)
cloning confidence! • Fast, with short extension times (10–30 s per kb)

PCR Polymerase Selection Chart for Cloning OneTaq®  DNA Polymerase

STANDARD HIGH-FIDELITY SPECIALTY • E xceptional performance across a wide range

PCR PCR PCR of templates (from high AT to high GC)
Long
Highest Fidelity Amplicons • H
 ot Start version allows room temperature
reaction setup; no activation step
OneTaq/ Taq / Phusion®(1) / LongAmp®/
OneTaq Hot Start Q5/ Q5 Phusion(1) LongAmp • Compatible with standard Taq protocols
Hot Start Taq Hot Start Flex Hot Start Taq
PROPERTIES
Fidelity vs. Taq 2X 1X ~280X(3) > 39X 2X TOOLS & RESOURCES
Amplicon Size < 6 kb ≤ 5 kb ≤ 20 kb ≤ 20 kb ≤ 30 kb
Extension Time 1 kb/min 1 kb/min 6 kb/min 4 kb/min 1.2 kb/min Visit NEBPCRPolymerases.com to find:
Resulting Ends 3´ A/Blunt 3´ A Blunt Blunt 3´ A/Blunt
3´→ 5´ exo Yes No Yes Yes Yes
• The full list of polymerases available
5´→ 3´ exo Yes Yes No No Yes • FAQs & troubleshooting guides
Units/50 µl Reaction 1.25 1.25 1.0 1.0 5.0
Annealing Temperature Tm–5 Tm–5 Tm+3 Tm+3 Tm–5 • Interactive tools to help with experimental design

APPLICATIONS • V ideos for setting up PCR reactions

Routine PCR ★ l l l l
• F or additional help with choosing the right polymerase for
Colony PCR ★ l
your PCR, we recommend using our PCR Selector at
Enhanced Fidelity l ★ l l
PCRSelector.neb.com.
High Fidelity ★ l

High Yield ★ l ★ l

Fast ★ l

Long Amplicon ★ l ★
GC-rich Targets ★ ★ l
 id you know that most high-
D
AT-rich Targets ★ l ★ l l fidelity polymerases benefit from
High Throughput l l l l
a Tm+3 annealing temperature?
Multiplex PCR l ★(2) l l

DNA Labeling ★
Use the NEB Tm Calculator at
Site-directed Mutagenesis ★ l TmCalculator.neb.com to
FORMATS ensure successful PCR.
Hot Start Available l l l l l

Kit l l l l

Master Mix Available l l l l l

Direct Gel Loading l l

(1) Phusion DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by
New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific.
(2) Use Multiplex PCR 5X Master Mix.
(3) Due to the very low frequency of misincorporation events being measured, the error rate of high-fidelity enzymes like Q5 is challenging to
measure in a statistically significant manner. We continue to investigate improved assays to characterize Q5's very low error rate to ensure that
we present the most robust accurate fidelity data possible (Popatov, V. and Ong, J.L. (2017) PLoS One, 12(1):e0169774. doi 10.1371/journal.
pone. 0169774).
Why choose
★ indicates recommended choice for application Q5 for your
PCR?

9
 cDNA SYNTHESIS

Ensure robust, full length cDNA synthesis – every time

NEB offers a selection of reverse transcriptases (RTs), which are available as RECOMMENDED PRODUCT
standalone enzymes or kits, to enable workflow customization. For difficult
targets or those with complex secondary structure, choose an RT with high ProtoScript® II First Strand
thermostability, as thermostable RTs are able to perform at higher tempera- cDNA Synthesis Kit (NEB #E6560)
tures on relaxed secondary structures. RTs with RNase H activity can degrade • H
 igh-quality, robust cDNA synthesis for a wide range
RNA:DNA hybrids, negatively impacting cDNA length and yield. Choosing of templates
an RT with reduced RNase H activity (RNase H–) increases yield, and results in
• Master mix formulation speeds reaction setup
more full-length cDNA product. Following reverse transcription, we recom-
mend using a high-fidelity DNA polymerase to generate double-stranded (ds) • Improved thermostability over standard M-MuLV
cDNA for cloning. Reverse Transcriptase

• Increased performance and value

cDNA Synthesis Selection Chart
• Also available as a standalone enzyme
cDNA SYNTHESIS FEATURES
KITS

• Ideal for cDNA synthesis in a two-step RT-qPCR workflow

LunaScript™ RT SuperMix Kit
(NEB #E3010)
ProtoScript® II First Strand
cDNA Synthesis Kit
(NEB #E6560)
ProtoScript First Strand
cDNA Synthesis Kit
(NEB #E6300)
Template Switching
RT Enzyme Mix
(NEB #M0466)
STANDALONE REAGENTS
• Single tube supermix contains random hexamer and
oligo-dT primers, dNTPs, Murine RNase Inhibitor, and
Luna Reverse Transcriptase
• Visible blue tracking dye for easy reaction setup
• Fast 13-minute protocol
• Generates cDNA at least 10 kb in length
• Contains ProtoScript II Reverse Transcriptase, an enzyme
with increased thermostability and reduced RNase H activity
• Convenient 2-tube kit includes dNTPs, Oligo-dT primer and
Random Primer Mix
• Generates cDNA at least 5 kb in length
• Contains M-MuLV Reverse Transcriptase
• Convenient 2-tube kit includes dNTPs, Oligo-dT primer and
Random Primer Mix
• Incorporates a universal adaptor sequence at the 3´ end of
cDNA during the RT reaction
• Enzyme mix and buffer are optimzed for efficient template
switching
• RT enzyme mix includes RNase Inhibitor
• High sensitivity for cDNA amplification – enables
transcriptome analysis by RNA-seq from single cells or as low
as 2 pg of human total RNA
• Robust and simple workflow for 5´ Rapid Amplification of
cDNA Ends (RACE)
• Retains the complete 5´ end of transcripts for 2nd Strand cDNA
Synthesis

ProtoScript II Reverse • RNase H– mutant of M-MuLV Reverse Transcriptase with

Transcriptase (NEB #M0368) increased thermostability and reduced RNase H activity
An alternative to SuperScript® II • Increased reaction temperatures (37–50°C)
M-MuLV Reverse Transcriptase • Robust reverse transcriptase for a variety of templates
(NEB #M0253) • Standard reaction temperatures (37–45°C)
• Robust reverse transcriptase for a broad temperature range
AMV Reverse Transcriptase (37–52°C)
(NEB #M0277) • Can be used for templates requiring higher
reaction temperatures
WarmStart RTx Reverse • Permits room temperature reaction setup
Transcription • Increased reaction temperatures (50–65°C)
(NEB #M0380) • Optimized for RT-LAMP isothermal detection
10
 DIGESTION

Experience the convenience of NEB restriction enzymes

Let NEB restriction enzymes bring convenience to your cloning workflow. With RECOMMENDED PRODUCTS
over 215 restriction enzymes that are 100% active in a single buffer, CutSmart®
Buffer, it is much simpler to set up your double digest reactions. Furthermore, High-Fidelity (HF®) Restriction Enzymes
you can speed up your reactions by choosing one of our Time-Saver™ qualified • O
 ne-buffer convenience with no loss of performance
restriction enzymes. These enzymes will digest DNA in 5–15 minutes, and can (CutSmart Buffer)
also be used safely overnight with no loss of sample. With over 230 specificities
• R educed star activity eliminates unwanted cleavage
to choose from, you can be sure to find the enzyme you need.
• T ime-Saver qualified for 5–15 minute digests or can digest
For the full list of restriction enzymes available from NEB, as well as the latest safely overnight without sample loss
activity/performance chart, visit NEBRestrictionEnzymes.com.
• E ngineered performance under a wide range
Competitor of conditions
NEB EcoRI-HF FastDigest® EcoRI
0 1 3 5 10 M 0 1 3 5 10 µl • A dded flexibility without added cost

• 6 X Gel-Loading Dye, Purple included with most

restriction enzymes and all HFs –sharpens bands and
leaves no UV shadow

Gel Loading Dye, Purple (6X) (NEB #B7024)

Unwanted
Cleavage
• B righter, sharper bands vs. glycerol based dyes
• No UV shadow, allowing for publication grade images

light blue

EcoRI-HF (NEB #R3101) shows no star activity in overnight digests, even when used at higher concentrations.
50 μl reactions were set up using 1 μg of Lambda DNA, the indicated amount of enzyme and the recommended
reaction buffer. Reactions were incubated overnight at 37°C. Marker M is the 1 kb DNA Ladder (NEB# N3232).
red/pink

NEB Restriction Enzymes by the Numbers Gel Loading Dye,

Purple (6X)
Gel Loading Dye,
Blue (6X)

286
216
Restriction enzymes COMPARISON OF DYE FRONTS
available from NEB

TOOLS & RESOURCES

CutSm Ar
Novel Application:
5C
4C
Chromosome Visit NEBRestrictionEnzymes.com to find:
conformation • The full list of restriction enzymes available
3C
capture enzymes • The latest activity/performance chart
Cutting to completion is critical to this application and NEB® offers These enzymes are 100% active in a single buffer, CutSmart®,
the largest selection of robust 4-, 5- and 6-bp cutters in the industry. making double digests so easy and convenient!
• V ideos for setting up restriction enzyme digests, double
35
HF all
Supplied with
digestions and troubleshooting reactions
Gel Loading Dye,

195 Time-Saver
Purple (6X)
enzymes Online interactive tools to help with setting up
enzymes With our High-Fidelity (HF®) restriction enzymes, you’ll see
reduced star activity. Rapidly digest your DNA (5–15 min.)
your restriction enzyme digests:
Digest 1 µg of your substrate DNA in 5–15 minutes using CutSmart Buffer. The included purple gel loading dye
using 1 µl of our Time-Saver™ qualified enzymes. sharpens bands and eliminates UV shadow. Enzyme Double Digest
You can also use these in overnight reactions.
Finder Finder

Thirteen 50 11
8
NEBcutter NEBioCalculator
® ®

5´ Type IIS 3´
-base
cutters 3´ enzymes 5´
enzymes
REBASE ®
NEBcloner
8-base cutters are known as rare-cutters – Type IIs enzymes cleave outside of their recognition Nicking enzymes “nick” one strand of the dsDNA,
they cut less often in a genome and can therefore sequence and are useful in DNA assembly methods, rather than both. They are being used in
be useful in your cloning experiments. including Golden Gate assembly. applications such as SDA and optical mapping.

11
 DNA END MODIFICATIONS

DNA end modifications that will improve your results

Make blunting fast and easy RECOMMENDED PRODUCT
Deciding which blunting enzyme to use depends on which activity you are looking
Quick Blunting™ Kit (NEB #E1201)
for: fill in of a 5´ overhang, removal of a 3´ overhang or removal of a 5´ overhang.
Once you have blunted your vector, a dephosphorylation step may still be necessary • O
 ptimized mix for blunting up to 5 µg of DNA in less than
to prevent self-ligation. 30 minutes

• R eactions are performed at room temperature in a

Blunting Selection Chart
ready-to-use mix
T4 DNA DNA Polymerase I, Quick Blunting Mung Bean
Polymerase* Large (Klenow) Kit Nuclease • C
 ontains T4 DNA Polymerase and T4 Polynucleotide
(NEB #M0203) Fragment (NEB #M0210) (NEB #E1201) (NEB #M0250) Kinase, enabling blunting and phosphorylation in one step
APPLICATION

Fill in of 5´ overhangs l l l

Removal of 3´ overhangs l l l l

Removal of 5´ overhangs l

* T4 DNA Polymerase has a strong 3´→ 5´ exo activity.

BLUNTING WITH DNA POLYMERASES

Reduce background with dephosphorylation RECOMMENDED PRODUCT

Did you know that it’s possible to reduce background from your cloning vector
Quick CIP (NEB #M0525)
by dephosphorylating your vector prior to ligation? A heat-labile phosphatase
with high specific activity is the best choice for dephosphorylation. This allows • Complete dephosphorylation in 10 minutes
you to add the enzyme directly to most buffers, quickly heat inactivate, and • R apid and irreversible heat inactivation
move on to the next step in your workflow. No need to purify. eliminates unwanted activity
• Faster reaction setup
Phosphatases Selection Chart (no supplemental additives like zinc required)
Recombinant Shrimp Antarctic
Alkaline Phosphatase Phosphatase (AP) Quick CIP • Flexible reaction conditions
(rSAP) (NEB #M0371) (NEB #M0289) (NEB #M0525) (active in any restriction enzyme buffer, no clean-up required)
FEATURES
100% heat inactivation 5 minutes/65°C 2 minutes/80°C 2 minutes/80°C • No need for multiple phosphatases (removes
High specific activity l l 5´- and 3´- phosphates from DNA, RNA and dNTPs )
Improved stability l l

Works directly in NEB buffers l l l

• Active on unincorporated dNTPs in PCR products –
Requires additive  l (Zn2+) improves DNA sequencing and SNP analysis
Quick Protocol l

Find an
overview of
dephosphorylation.

12
 DNA END MODIFICATIONS

Easily generate dA-tails for PCR cloning

Tailing is typically done to prepare a T-vector for use in TA cloning or to
RECOMMENDED PRODUCT
dA-tail a PCR product produced by a high-fidelity polymerase (not Taq DNA
Polymerase) for use in TA cloning. Klenow Fragment (3´→5´ exo-) (NEB #M0212)

Tailing Selection Chart • F ully active in NEBuffers 1.1, 2.1, 3.1 and CutSmart (when
supplemented with dNTPs), which eliminates the need to
Klenow Fragment (3´→5´ exo-) clean up the reaction for downstream applications
(NEB #M0212) Taq DNA Polymerase
FEATURES
• H
 eat inactivated at 75° C after 20 minutes

Reaction temperature 37°C 75°C

Heat inactivated 75°C, 20 minutes No
Nucleotide cofactor dATP dATP

LEARN MORE ABOUT PCR CLONING

When is a phosphorylation step needed? RECOMMENDED PRODUCT

Typically, PCR primers are not phosphorylated; the 5´ ends of the amplicon need
to be treated by a kinase to introduce the 5´ phosphate necessary for ligation, if
cloning into a dephosphorylated vector.
T4 Polynucleotide Kinase (NEB #M0201)
• H
 ighly-efficient catalysis of the 5´ addition of Pi to ds- &
ssDNA, RNA, as well as nucleoside 3´ monophosphates

THE MECHANISM OF DNA PHOSPHORYLATION

Featured Online Tool: NEBCloner

Use NEBcloner at NEBcloner.neb.
com to find the right products and
protocols for each step in your traditional
cloning experiment. Also find other
relevent tools and resources to enable
protocol optimization.

13
 LIGATION

Easy & effective ligation for all end types

Once your insert and vector are end-treated, as needed, and have compatible ends,
RECOMMENDED PRODUCTS
you’ll be ready to ligate. NEB offers an extensive selection of high-quality and per-
formance-optimized DNA ligases and ligase master mixes; you’ll be certain to find • F or the ligation of sticky ends, NEB recommends
an enzyme specific for your end type, enabling maximum efficiency. Since time is our Instant Sticky-end Ligase Master Mix
always at a premium in the lab, you can speed up your reaction setup and incubation (NEB #M0370) (no incubation needed) or the
by choosing one of our ligase master mixes. With so many ligases to choose from, Quick Ligation™ Kit (NEB #M2200)
make NEB your source for DNA ligases. • F or the ligation of blunt ends, NEB recommends our
Blunt/TA Ligase Master Mix (NEB #M0367) or
DNA Ligase Selection Chart for Cloning the Quick Ligation Kit (NEB #M2200)
• T he Blunt/TA Ligase Master Mix (NEB #M0367)
Blunt/TA Ligase Master Mix
Master Mix (NEB #M0370)
Instant Sticky-end Ligase

is recommended for T/A cloning

HiFi Taq DNA Ligase

Quick Ligation Kit
ElectroLigase®

T4 DNA Ligase

T3 DNA Ligase

T7 DNA Ligase
TOOLS & RESOURCES
(NEB #M0367)

(NEB #M0369)

(NEB #M0202)

(NEB #M2200)

(NEB #M0317)

(NEB #M0318)

(NEB #M0647)
Visit NEBStickTogether.com to find:
• T he full list of ligases available
DNA APPLICATIONS
• F AQs
Ligation of sticky ends lll ll ll ll lll ll ll l
• V ideos about ligation and help with setting up
ligation reactions
Ligation of blunt ends l lll ll ll lll ll
Learn more about NEB's research on ligase fidelity and
how it enables improved DNA assembly methods (such as
T/A cloning l lll ll ll ll l l Golden Gate) at www.neb.com/GoldenGate

Online interactive tools to help with setting up

Electroporation lll ll
your restriction enzyme digests:

Ligation of sticky ends only lll

NEBcloner NEBioCalculator
Repair of nicks
ll ll ll lll ll ll ll ll
in dsDNA

High complexity
ll ll ll lll ll
library cloning

FEATURES

Salt tolerance ( > 2X that

of T4 DNA Ligase) 

Ligation in 15 min. or less       

Master Mix Formulation  

Thermostable 

Recombinant        

KEY
lll Recommended product(s) l Will perform selected application, ll Works well for
for selected application but is not recommended selected application

Find an
overview of
ligation.

14
 TRANSFORMATION

Maximize your transformation efficiency

Selecting a competent cell line for cloning requires that you strike the right RECOMMENDED PRODUCTS
balance; you want a strain that offers high transformation efficiencies, but also
has the ability to generate sufficient plasmid yields. NEB cloning strains offer NEB 5-alpha Competent E.coli (NEB #C2987)
high transformation efficiencies, and are T1 phage resistant and endA deficient,
• C
 loning strain with same genetic features as the
ensuring high-quality plasmid preparation. Additionally, RecA– strains minimize popular DH5α™
potential plasmid recombination. For convenience, NEB cloning strains are
available in a variety of formats, including single-use tubes, 96-well plate, • H
 igh transformation efficiencies, available as
electrocompetent or chemically competent
384-well plate, and strip tubes. Electrocompetent and subcloning efficiencies are
also available. Your choice of competent cell line for cloning can influence the • C
 onvenient formats, including single-use tubes,
success of your cloning experiment; NEB offers a cell line for every need. 96-well plate, 384-well plate, and strip tubes

• Value pricing
Competent Cell Selection Chart
• RecA– strain
NEB 5-alpha
NEB 5-alpha NEB Turbo F´ I q NEB 10-beta dam –/dcm – NEB Stable
Competent Competent Competent Competent Competent Competent NEB Cloning Competent E.coli Sampler
E. coli E. coli E. coli E. coli E. coli E. coli (NEB #C1010)
(NEB #C2987) (NEB #C2984) (NEB #C2992) (NEB #C3019) (NEB #C2925) (NEB #C3040)
FEATURES • C
 ontains our four most popular cloning strains:
Versatile l l
NEB 5-alpha, NEB 10-beta, NEB Turbo and NEB Stable
Competent E. coli
Fast growth l
(< 8 hours)
Toxic gene cloning l l l TOOLS & RESOURCES
Large plasmid/BAC
cloning
l l
Visit www.neb.com/CloningCompCells
Dam/Dcm free
to find:
l
plasmid growth • T he full list of competent cells available
Retroviral/lentiviral
l • F AQs
vector cloning
recA- l l l l
• Technical tips and troubleshooting guides

endA- l l l l l l • P rotocol and optimization videos

FORMATS

Chemically
l l l l l l
Competent
Electrocompetent l l l

Subcloning l

96-well format* l

384-well format* l

12 x 8 tube strips* l THE MECHANISM OF TRANSFORMATION WITH

*Other strains are available on request. for more information contact custom@neb.com. COMPETENT CELLS

Benefit from High Transformation Efficiencies

NO DRY ICE CHARGES
Transformation Efficiency (x109)

2.5

2.0
with Competent Cells from NEB
(cfu/µg pUC19)

1.5

1.0

0.5

0
®
®
®
®

le
a

o
a
ph

3™ t
h1 ot
rb

ab
H ic ot
et
1 y

bl ho
y
-T nc

B ™ nc

ac h
D ff h
-b

Tu
al

St

St S
M S
R

S
™ e

10 ie
5-

10
5α ci

EB

ne
ne

Find tips
®

EB
AX ne
H ffi

EB
EB

O
O
M O

N
D XE

N
E
N

for successful
N

A
M

transformation.
The transformation efficiencies were compared using manufacturers’ recommended protocols.
Values shown are the average of triplicate experiments.
15
 ANALYSIS

Take DNA analysis to the next level

NEB’s DNA ladders help to take the guesswork out of your sample analysis during gel electrophoresis.
With sharp, evenly spaced reference bands that are easy to identify, our DNA Ladders can be used for
sample quantitation. Choose the DNA ladder that best suits your target insert and vector sizes. Try our
Quick-Load Purple DNA Ladders, which include a vial of our Purple Gel Loading Dye (no SDS), for
sharper bands and no UV shadow.

Quick-Load and Quick-Load Purple DNA Ladders

kb
40.0
20.0
kb 15.0
10.0 bp
kb bp
8.0 1,350 10.0
10.0 766
6.0
5.0 8.0 8.0
4.0 6.0 916
3.0 766 6.0
5.0 500
bp 700 5.0
650
1,517 4.0 600
2.0 550 4.0
350
500
1,200 450 300
1.5 3.0
400 250 3.0
1.2 1,000 350
900 200
1.0 2.0 300
800
0.9 250 150 2.0
0.8 700
0.7 600 1.5 200
0.6 100 1.5
500/517
0.5 150 75
400
0.4 1.0
300 50 1.0
0.3 100

200
0.2
25
100
0.5 50
0.1 0.5
1 kb Plus DNA Ladder*,*** 100 bp DNA Ladder*,*** 1 kb DNA Ladder*,*** 50 bp DNA Ladder*** Low Molecular Weight Quick-Load 1 kb Extend DNA Ladder
(NEB #N3200) (NEB #N3231) (NEB #N3232) (NEB #N3236) DNA Ladder*** (NEB #N3239) 0.6% TBE agarose gel.
1.0% TBE agarose gel. 1.3% TAE agarose gel. 0.8% TAE agarose gel. 3.0% TBE agarose gel. (NEB #N3233) 3.0% TBE agarose gel. Mass values are for 0.5 µg/lane.
Quick-Load Purple Format Quick-Load Purple Format Quick-Load Purple Format Quick-Load Purple Format Quick-Load Purple Format
(NEB #N0550) (NEB #N0551) (NEB #N0552) (NEB #N0556) (NEB #N0557)

* Available in Quick-Load® and TriDye™ formats Ready-to-Load ***  Free Loading Dye included

Additional DNA Ladders from NEB

NEW RECOMMENDED PRODUCTS
bp
700
650
1 kb Plus DNA Ladder for Safe Stains
400
(NEB #N0559)
300
TriDye™ Ultra Low Range DNA Ladder
200 Can be used with E-Gels®
(NEB #N0558)
150 kb
NEW bp 10.0
Quick-Load® Purple 1 kb Plus, 100 bp, 1 kb,
766 5.0
100

75
3.0 50 bp and Low Molecular Weight DNA Ladders
kb
10.0 500
2.0 (NEB #N0550, #N0551, #N0552, #N0556, #N0557)
50 8.0
6.0 1.5
5.0
4.0
• Ideal for analysis of DNA of various sizes
35 3.0 1.0
2.0 300
0.766
• Convenient, ready-to-load format
1.5
25 1.2
1.0 0.500
• Sharp, high contrast bands
0.9
0.8
0.7
150
• U
 tilizes purple gel loading dye which is also supplied with
0.6 0.300
0.5 HF restriction enzymes
15 0.4

0.3
0.150
• Includes vial of purple dye (no SDS) (NEB #B7025)
0.2 0.050

0.1
50
10

TriDye Ultra Low Range

™ 1 kb Plus DNA Ladder PCR Marker*** Fast DNA Ladder
DNA Ladder for Safe Stains (NEB #N3234) (NEB #N3238)
(NEB #N0558) (NEB #N0559) 1.8% TBE agarose gel. 1.2% TBE agarose gel.
16 20% polyacrylamide gel. 1.0% TBE agarose gel.
 MONARCH NUCLEIC ACID PURIFICATION

Monarch® Nucleic Acid Purification Kits

The need for high quality, highly pure DNA and RNA is important for many RECOMMENDED PRODUCTS
molecular cloning workflows. These nucleic acids are being purified from a
wide variety of sources, such as cells and tissues, enzymatic reactions (e.g., Monarch Plasmid Miniprep Kit
PCR, ligation, digestions), and agarose gel matrices, to name a few. Purification (NEB #T1010)
methods have been, and continue to be, optimized for various starting Monarch DNA Gel Extraction Kit
materials to ensure excellent recovery, high purity, minimal processing time and (NEB #T1020)
compatibility with emerging techniques. The Monarch Nucleic Acid Purification Monarch PCR & DNA Cleanup Kit
product portfolio addresses the needs of researchers upstream and downstream (NEB #T1030)
of their molecular cloning workflows, including products for isolation of DNA
Monarch Genomic DNA Purification Kit
and RNA from biological samples, DNA and RNA cleanup, plasmid purification (NEB #T3010)
and gel extraction.
Monarch Total RNA Miniprep Kit
(NEB #T2010)

Monarch Nucleic Acid Purification Kits

TOOLS & RESOURCES
PRODUCT APPLICATIONS FEATURES

Monarch Plasmid Purification of up to • E lute in as little as 30 µl Visit NEBMonarch.com to find:

Miniprep Kit 20 µg of plasmid DNA • P revent buffer retention and salt carryover
(NEB #T1010) from bacterial culture. with optimized column design • Protocol videos and optimization tips
• Includes
 colored buffers to monitor • Troubleshooting guide & FAQs
completion of certain steps
• N o need to add RNase before starting • Tips for recycling your Monarch kit

Monarch DNA Gel Purification of up to • E lute in as little as 6 µl

Extraction Kit 5 µg of DNA from • P revent buffer retention and salt carryover
(NEB #T1020) agarose gels. with optimized column design
• F ast, user-friendly protocol

Monarch PCR & DNA Purification and • E lute in as little as 6 µl

Cleanup Kit concentration of up • P revent buffer retention and salt carryover
(NEB #T1030) to 5 µg of DNA from with optimized column design
enzymatic reactions. • P urify oligos and other small DNA fragments
with simple protocol modification
TIPS FOR RECYCLING YOUR MONARCH KIT
Monarch Genomic Extraction and • O ptimized protocols and buffer chemistry for
DNA Purification Kit purification of genomic excellent yields
(NEB #T3010) DNA from cells, blood, • P urifies gDNA with a peak size > 50 kb
tissues and other Designed for performance
• Includes RNase A and Proteinase K
sample types.
• P rotocol also available for gDNA cleanup
NEB Qiagen
Monarch Total RNA Extraction and • P urifies RNA of all sizes, including miRNA
Miniprep Kit purification of up to & small RNAs > 20 nucleotides Buffer
(NEB #T2010) 100 µg of total RNA • Includes DNase I, gDNA removal columns, retention
from blood, cells, Proteinase K, and a stabilization reagent
tissues and other • P rotocols also available for RNA
sample types. fractionation and RNA cleanup

Visit NEBMonarch.com
to learn more and access
supporting content.

17
 ORDERING INFORMATION

Selected Products for PCR & Mutagenesis Products for cDNA Synthesis
PRODUCT NEB # SIZE PRODUCT NEB # SIZE

HIGH-FIDELITY DNA POLYMERASES AMV Reverse Transcriptase M0277S/L 200/1,000 units

LunaScript™ RT SuperMix Kit E3010S/L 25/100 reactions
Q5 High-Fidelity DNA Polymerase M0491S/L 100/500 units
M-MuLV Reverse Transcriptase M0253S/L 10,000/50,000 units
Q5 Hot Start High-Fidelity
M0493S/L 100/500 units ProtoScript II First Strand
DNA Polymerase E6560S/L 30/150 reactions
cDNA Synthesis Kit
Q5 High-Fidelity 2X Master Mix M0492S/L 100/500 reactions
ProtoScript First Strand
Q5 Hot Start High-Fidelity E6300S/L 30/150 reactions
M0494S/L 100/500 reactions cDNA Synthesis Kit
2X Master Mix
Template Switching RT Enzyme Mix M0466S/L 20/100 reactions
Q5 High-Fidelity PCR Kit E0555S/L 50/200 reactions
ProtoScript II Reverse Transcriptase M0368S/L/X 4,000/10,000/40,000 units
Phusion High-Fidelity PCR Master Mix
M0531S/L 100/500 reactions WarmStart® RTx Reverse Transcriptase M0380S/L 50/250 reactions
with HF Buffer
Phusion High-Fidelity PCR Master Mix
M0532S/L 100/500 reactions
with GC Buffer
Products for Restriction Digestion
Phusion Hot Start Flex 2X Master Mix M0536S/L 100/500 reactions
Phusion High-Fidelity PCR Kit E0553S/L 50/200 reactions PRODUCT NEB # SIZE
Phusion High-Fidelity HIGH-FIDELITY (HF ) RESTRICTION ENZYMES
®
M0530S/L 100/500 units
DNA Polymerase
AgeI-HF R3552S/L 300/1,500 units
Phusion Hot Start Flex
M0535S/L 100/500 units ApoI-HF R3566S/L 1,000/5,000 units
High-Fidelity DNA Polymerase
BamHI-HF R3136S/L/T/M 10,000/50,000 units
DNA POLYMERASES
BbsI-HF R3539S/L 300/1,500 units
OneTaq DNA Polymerase M0480S/L/X 200/1,000/5,000 units
BclI-HF R3160S/L 3,000/15,000 units
OneTaq Hot Start DNA Polymerase M0481S/L/X 200/1,000/5,000 units
BmtI-HF R3658S/L 300/1,500 units
OneTaq 2X Master Mix
M0482S/L 100/500 reactions BsaI-HFv2 R3733S/L 1,000/5,000 units
with Standard Buffer
OneTaq Quick-Load 2X Master Mix BsiWI-HF R3553S/L 300/1,500 units
M0486S/L 100/500 reactions
with Standard Buffer BsrFI-v2 R0682S 1,000 units
OneTaq Hot Start 2X Master Mix BsrGI-HF R3575S/L 1,000/5,000 units
M0484S/L 100/500 reactions
with Standard Buffer BstEII-HF R3162S/L/M 2,000/10,000 units
OneTaq Hot Start 2X Master Mix BstZ171-HF R3594S/L 1,000/5,000 units
M0485S/L 100/500 reactions
with GC Buffer
DraIII-HF R3510S/L 1,000/5,000 units
OneTaq Hot Start Quick-Load
M0488S/L 100/500 reactions EagI-HF R3505S/L/M 500/2,500 units
2X Master Mix with Standard Buffer
EcoRI-HF R3101S/L/T/M 10,000/50,000 units
OneTaq Hot Start Quick-Load
M0489S/L 100/500 reactions EcoRV-HF R3195S/L/T/M 4,000/20,000 units
2X Master Mix with GC Buffer
Taq DNA Polymerase with HindIII-HF R3104S/L/T/M 10,000/50,000 units
M0267S/L/X/E 400/2,000/4,000/20,000 units
ThermoPol™ Buffer KpnI-HF R3142S/L/M 4,000/20,000 units
Taq DNA Polymerase with MfeI-HF R3589S/L 500/2,500 units
M0273S/L/X 400/2,000/4,000 units
Standard Taq Buffer
MluI-HF R3198S/L 1,000/5,000 units
Taq DNA Polymerase with
M0320S/L 400/2,000 units NcoI-HF R3193S/L/M 1,000/5,000 units
Standard Taq (Mg-free) Buffer
NheI-HF R3131S/L/M 1,000/5,000 units
Taq PCR Kit E5000S 200 reactions
NotI-HF R3189S/L/M 500/2,500 units
Quick-Load Taq 2X Master Mix M0271L 500 reactions
NruI-HF R3192S/L 1,000/5,000 units
Taq 2X Master Mix M0270L 500 reactions
NsiI-HF R3127S/L 1,000/5,000 units
Taq 5X Master Mix M0285L 500 reactions
PstI-HF R3140S/L/T/M 10,000/50,000 units
Multiplex PCR 5X Master Mix M0284S 100 reactions
PvuI-HF R3150S/L 500/2,500 units
Hot Start Taq DNA Polymerase M0495S/L 200/1,000 units
PvuII-HF R3151S/L/M 5,000/25,000 units
Hot Start Taq 2X Master Mix M0496S/L 100/500 reactions
SacI-HF R3156S/L/M 2,000/10,000 units
Vent DNA Polymerase M0254S/L 200/1,000 units
SalI-HF R3138S/L/T/M 2,000/10,000 units
Vent (exo ) DNA Polymerase
–
M0257S/L 200/1,000 units
SbfI-HF R3642S/L 500/2,500 units
Deep Vent DNA Polymerase M0258S/L 200/1,000 units
ScaI-HF R3122S/L/M 1,000/5,000 units
Deep Vent (exo–) DNA Polymerase M0259S/L 200/1,000 units
SpeI-HF R3133S/L/M 500/2,500 units
LongAmp Taq DNA Polymerase M0323S/L 500/2,500 units
SphI-HF R3182S/L/M 500/2,500 units
LongAmp Hot Start Taq
M0534S/L 500/2,500 units SspI-HF R3132S/L/M 1,000/5,000 units
DNA Polymerase
StyI-HF R3500S/L 3,000/15,000 units
LongAmp Taq 2X Master Mix M0287S/L 100/500 reactions
OTHER POPULAR RESTRICTION ENZYMES
LongAmp Hot Start Taq 2X Master Mix M0533S/L 100/500 reactions
LongAmp Taq PCR Kit E5200S 100 reactions AscI R0558S/L 500/2,500 units
AvrII R0174S/L 100/500 units
PCR CLONING & MUTAGENESIS
BglII R0144S/L/M 2,000/10,000 units
NEB PCR Cloning Kit E1202S 20 reactions
BsaI R0535S/L 1,000/5,000 units
NEB PCR Cloning Kit
E1203S 20 reactions BsmBI R0580S/L 200/1,000 units
(Without Competent Cells)
DpnI R0176S/L 1,000/5,000 units
Q5 Site-Directed Mutagenesis Kit E0554S 10 reactions
Esp3I R0734S/L 300/1,500 units
Q5 Site-Directed Mutagenesis Kit
MluI R0198S/L 1,000/5,000 units
(Without Competent Cells) E0552S 10 reactions
NcoI R0193S/L/T/M 1,000/5,000 units
KLD Enzyme Mix M0554S 25 reactions
NdeII R0111S/L 4,000/20,000 units
Deoxynucleotide (dNTP) Solution Set N0446S 25 μmol of each
NheI R0131S/L/M 1,000/5,000 units
18 Deoxynucleotide (dNTP) Solution Mix N0447S/L 8/40 μmol of each
 ORDERING INFORMATION

Products for Restriction Digestion (Cont.) Products for Nucleic Acid Purification
PRODUCT NEB # SIZE PRODUCT NEB # SIZE
OTHER POPULAR RESTRICTION ENZYMES (CONT'D) Monarch Plasmid Miniprep Kit T1010S/L 50/250 preps
PacI R0547S/L 250/1,250 units Monarch DNA Gel Extraction Kit T1020S/L 50/250 preps
PmeI R0560S/L 500/2,500 units Monarch PCR & DNA Cleanup Kit (5 μg) T1030S/L 50/250 preps
SmaI R0141S/L 2,000/10,000 units Monarch Genomic DNA Purification Kit T3010S/L 50/150 preps
SpeI R0133S/L/M 500/2,500 units Monarch Total RNA Miniprep Kit T2010S 50 preps
XhoI R0146S/L/M 5,000/25,000 units Columns and buffers also available separately.
XbaI R0145S/L/T/M 3,000/15,000 units
XmaI R0180S/L/M 500/2,500 units Products for DNA Analysis
FEATURED GEL LOADING DYE
PRODUCT NEB # SIZE
Gel Loading Dye, Purple (6X) B7024S 4 ml
1 kb DNA Ladder N3232S/L 200/1,000 gel lanes
Gel Loading Dye, Purple (6X), no SDS B7025S 4 ml
TriDye 1 kb DNA Ladder N3272S 125 gel lanes
For the full list of restriction enzymes available, visit www.neb.com.
Quick-Load 1 kb DNA Ladder N0468S/L 125/375 gel lanes
100 bp DNA Ladder N3231S/L 100/500 gel lanes
Products for End Modification
TriDye 100 bp DNA Ladder N3271S 125 gel lanes
PRODUCT NEB # SIZE Quick-Load 100 bp DNA Ladder N0467S/L 125/375 gel lanes
Quick CIP M0525S/L 1,000/5,000 units 1 kb Plus DNA Ladder N3200S/L 200/1,000 gel lanes
Shrimp Alkaline Phosphatase 1 kb Plus DNA Ladder for Safe Stains N0559S 50 µg/ml
M0371S/L 500/2,500 units
(Recombinant)
TriDye 1 kb Plus DNA Ladder N3270S 250 gel lanes
Antarctic Phosphatase M0289S/L 1,000/5,000 units
Quick-Load 1 kb Plus DNA Ladder N0469S 250 gel lanes
T4 DNA Polymerase M0203S/L 150/750 units
DNA Polymerase I, Quick-Load Purple 1 kb Plus DNA Ladder N0550S/L 250/750 gel lanes
M0210S/L/M 200/1,000/1,000 units
Large (Klenow) Fragment TriDye Ultra Low Range DNA Ladder N0558S 100 µg/ml
Quick Blunting Kit E1201S/L 20/100 reactions 50 bp DNA Ladder N3236S/L 200/1,000 gel lanes
Mung Bean Nuclease M0250S/L 1,000/5,000 units Quick-Load Purple 50 bp DNA Ladder N0556S 250 gel lanes
T4 Polynucleotide Kinase M0201S/L 500/2,500 units Quick-Load 1 kb Extend DNA Ladder N3239S 125 gel lanes
Klenow Fragment (3´ → 5´ exo–) M0212S/L/M 200/1,000/1,000 units
Quick-Load Purple 1 kb DNA Ladder N0552S/L 125/375 gel lanes
β-Agarase I M0392S/L 100/500 units
Quick-Load Purple 100 bp DNA Ladder N0551S/L 125/375 gel lanes
Low Molecular Weight DNA Ladder N3233S/L 100/500 gel lanes
Products for Ligation
Quick-Load Purple Low Molecular Weight
N0557S 125 gel lanes
PRODUCT NEB # SIZE DNA Ladder
Blunt/TA Ligase Master Mix M0367S/L 50/250 reactions Fast DNA Ladder N3238S 200 gel lanes
Instant Sticky-End Ligase Master Mix M0370S/L 50/250 reactions PCR Marker N3234S/L 100/500 gel lanes
ElectroLigase M0369S 50 reactions
T4 DNA Ligase M0202S/L/T/M 20,000/100,000 units Products for Seamless Cloning
Quick Ligation Kit M2200S/L 30/150 reactions
PRODUCT NEB # SIZE
T3 DNA Ligase M0317S/L 100,000/750,000 units
NEBuilder HiFi DNA Assembly Cloning Kit E5520S 10 reactions
T7 DNA Ligase M0318S/L 100,000/750,000 units
NEBuilder HiFi DNA Assembly Master Mix E2621S/L 10/50 reactions
Taq DNA Ligase M0208S/L 2,000/10,000 units
NEBuilder HiFi DNA Assembly Bundle
E2623S 20 reactions
for Large Fragments
Products for Transformation Gibson Assembly Cloning Kit E5510S 10 reactions
PRODUCT NEB # SIZE Gibson Assembly Master Mix E2611S/L 10/50 reactions
NEB Golden Gate Assembly Mix E1601S/L 20/100 reactions
20 x 0.05 ml/tube/
dam-/dcm- Competent E. coli C2925H/I BioBrick® Assembly Kit E0546S 50 reactions
6 x 0.2 ml ml/tube
20 x 0.05 ml/tube/ BbsI R0539S/L 300/1,500 units
6 x 0.2 ml/tube/ BbsI-HF R3539S/L 300/1,500 units
NEB 5-alpha Competent E. coli
C2987H/I/P/R/U 1 x 96 well plate/
(High Efficiency) BsaI R0535S/L 1,000/5,000 units
1 x 384 well plate/
12 x 8 tube strips BsaI-HFv2 R3733S/L 1,000/5,000 units
NEB 5-alpha Competent E. coli BsmBI R0580S/L 200/1,000 units
C2988J 6 x 0.4 ml/tube
(Subcloning Efficiency) Esp3I R0734S/L 300/1,500 units
NEB 5-alpha Electrocompetent E. coli C2989K 6 x 0.1 ml/tube T4 DNA Polymerase M0203S/L 150/750 units
NEB 5-alpha F´ Iq Competent E. coli Taq DNA Ligase M0208S/L 2,000/10,000 units
C2992H/I 20 x 0.05/6 x 0.2 ml
(High Efficiency)
T4 DNA Ligase M0202S/L/T/M 20,000/100,000 units
NEB 10-beta Competent E. coli 20 x 0.05 ml/tube/
C3019H/I T5 Exonuclease M0363S/L 1,000/5,000 units
(High Efficiency) 6 x 0.2 ml ml/tube
Exonuclease V (RecBCD) M0345S/L 1,000/5,000 units
NEB 10-beta Electrocompetent E. coli C3020K 6 x 0.1 ml/tube
USER Enzyme M5505S/L 50/250 units
NEB Turbo Competent E. coli 20 x 0.05 ml/tube/
C2984H/I Thermolabile USER Enzyme II M5508S/L 50/250 units
(High Efficiency) 6 x 0.2 ml/tube
NEB Turbo Electrocompetent E. coli C2986K 6 x 0.1 ml/tube
NEB Stable Competent E. coli
C3040H/I
20 x 0.05 ml/tube/ Products for Recombinational Cloning
(High Efficiency) 6 x 0.2 ml/tube
PRODUCT NEB # SIZE
NEB Cloning Competent E. coli Sampler C1010S 8 tubes
Cre Recombinase M0298S/L/M 50/250 units
For the full list of competent cells available, visit www.neb.com.
19
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One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For
more information, please email us at gbd@neb.com. The use of these products may require you to obtain additional third party intellectual
property rights for certain applications.
Your purchase, acceptance, and/or payment of and for NEB’s products is pursuant to NEB’s Terms of Sale at www.neb.com/support/terms-
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GIBSON ASSEMBLY® is a registered trademark of Synthetic Genomics, Inc. ONESHOT® and MAX EFFICIENCY® are registered trademarks
of Invitrogen. E-GELS®, FAST DIGEST® and PHUSION® are registered trademarks of Thermo Fisher Scientific. QIAQUICK® and MINELUTE®
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