NEB EXPRESSIONS
®
A scientific update from New England Biolabs®
Programming Life: Inquiry
& Engineering through
Synthetic Biology page 3
Golden Gate Assembly
page 8
NEBuilder® HiFi DNA Assembly
page 9
15 Inspiring Scientists Receive
Passion in Science Awards™ page 7
40th Anniversary of NEB
– The lucky winners page 6
Issue I, 2015
On the Shoulders of Giants
Bioinformatics
Sequencing Data
Cost of DNA Synthesis
Helping to Establish the Field of Synthetic Biology
Collaboration of biologists
and computer scientists led to
development of software tools for
biologists, including Basic Local
Alignment Search Tool (BLAST),
as well as other alignment and
codon optimization tools (4).
Automated DNA sequencers
became widespread and
more affordable, and the
first complete genome
of an organism was
sequenced (47–49).
Cost of synthesizing DNA dropped
1,000-fold in a decade, and speed
of production increased (50).
NEB receives
LIFE SCIENCE
INDUSTRY
AWARD for
“Best Molecular
Biology Products”
Custom Oligonucleotides
Recombinant DNA
In the late 1950s, Khorana
developed the synthetic approach
of blocking/deblocking cycles for
the stepwise elongation of oligos.
This eventually led to solid phase
synthesis and automation.
In 1972, the first published
report of recombinant DNA
made in vitro, and then
transformed into E. coli (42,43).
2010s
1990s
Contents
1970s
1950s
Issue I, 2015
2000s
3 Programming Life: Inquiry & Engineering
feature article
1980s
1960s
Gene Regulation
Polymerase chain
reaction (PCR)
Jacob & Monod first described a genetic
circuit (39), earning a Nobel prize in
1965. Seminal discoveries included the
lysis vs. lysogeny developmental switch
in bacteriophage lambda (λ) (40,41).
First uses of PCR in 1983
(44) helped fuel an explosion
of scientific applications,
from genetic engineering
to forensic science.
Early DNA Assembly
Genome Editing
One-Step DNA Assembly
First reports connecting
synthetic DNA to genome
editing in the lambda red
system in eubacteria. Later
developments include the
use of engineered zinc finger
nucleases, TALENS (29) and
CRISPR/Cas9 (30,31).
Development of one-step
assembly methods,
including Golden Gate (51),
USER®, Gibson Assembly®
(52) and NEBuilder HiFi
DNA Assembly (53).
Through Synthetic Biology
In vitro enzymatic approaches
using complementary,
overlapping oligonucleotides
enabled genes to be synthesized
directly from sequence (45,46).
Understanding the origins of this new and exciting field is just the
first step towards creating novel biological systems.
6
40th
anniversary
Prize Draw
Which was your first NEB catalog?
See what researchers in Europe shared regarding their first NEB
catalog and how they started their carreer
www.lifescienceindustryawards.com
7
BsaI
3´
15 Inspiring Scientists Receive
Passion in Science Awards
NEB celebrated its 40th anniversary by honoring the unsung heroes
of the laboratory for their shared values and vision.
BsaI
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P6
PCR amplification of
vector and fragments
Enjoy the single-tube simplicity of this straightforward, BsaI-based
assembly technique.
A
Single-tube reaction
• BsaI
• DNA ligase
PCRlinearized
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+
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PCR-amplified
fragments
9
new products
NEBuilder HiFi DNA Assembly
The next generation of DNA assembly and cloning
has arrived.
Primer
10 Rapid PCR Cleanup Enzyme Set
PCR product
new products
dNTPs
+ Rapid PCR Cleanup Enzyme Set
(1 µl of each enzyme per 5 µl of
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Incubate at 37°C
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Order your copy
of the upcoming
NEB CATALOG
& TECHNICAL
REFERENCE
2015/16
at your local
distributor!
Test now and order „Trial Size“ ...or larger!
11 Rapid PNGase F
Cleaned up
PCR product
new products
Quickly release N-linked oligosaccharides from your glycoprotein.
SNAP-Cell® 647-SiR
New fluorescent probe for intracellular live-cell imaging
12 Quick-Load Purple DNA Ladders
new products
new purple loading dye for sharper bands and no UV shadow
cover photo
Common Grape Hyacinth (Muscari neglectum) growing on the NEB campus.
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are registered trademarks of New England Biolabs, Inc.
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of New England Biolabs, Inc.
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TRACKIT™ is a trademark of Invitrogen™. Invitrogen™ is a trademark of
Life Technologies, Inc.
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feature article
Programming Life: Inquiry & Engineering Through
Synthetic Biology
The report of the first chimeric DNA molecule in 1968 (1) ushered in a new age for experimental biology and biotechnology. The
ability to propagate DNA obtained, in principle, from virtually any organism within the cytoplasm of Escherichia coli (2) set the stage for
sequencing of genes and genomes. This advance enabled researchers not only to connect a mutant phenotype with the corresponding
genotype, but also paved the way for the industrial production of medically important proteins such as insulin. The in vitro construction
of recombinant DNA thus became a cornerstone method in the functional and biochemical characterization of genes and proteins.
The five decades following the birth of molecular cloning have witnessed an incredible scaling-up of molecular biology due, in large
part, to the development of high-throughput technologies in nucleic acid sequencing and macromolecular analysis. But long absent from
the resulting explosion of information has been the ability to rationally recreate, in the laboratory, the regulatory complexity of the very
gene networks forming the basis cellular behavior. In short, we know a great deal about the “code of life” but are only now beginning
to be able to program with it. This aspiration has, in part, given birth to the rapidly developing field of synthetic biology, which aims to
unite the rigor of engineering with the design and construction of recombinant nucleic acids, with which to study and understand the
behavior of genetic circuits as well as utilize them for technological ends.
Peter Weigle, Ph.D., New England Biolabs, Inc.
and Laura Fulford, BiteSize Bio
What is Synthetic Biology?
Though a comprehensive definition of synthetic
biology is elusive, one may characterize it as a
“build to understand” approach to biology (3). A
quote by the famous theoretical physicist Richard
Feynman epitomizes a theme characteristic of the
field – “What I cannot create, I do not understand.” How does this sentiment relate to recombinant DNA? Imagine beginning with a repertoire
of well-characterized DNA “parts” encoding
biological functions such as receptors, promoters,
activators, repressors, terminators and reporter
genes (or other outputs) – and attempting to rearrange them into configurations designed to direct
a biological system (typically, a cellular “chassis”)
to accomplish a desired task. Think a pollution
detecting E. coli cell that expresses green fluorescent protein (GFP) in the presence of arsenic and
then self-destructs after a given period of time, or
an engineered implantable human cell line that
undergoes a preset number of cell divisions and
then secretes insulin at precisely regulated levels
in response to extracellular glucose concentrations.
Such re-purposed cells would be described, in
synthetic biology parlance, as “genetic devices.”
These devices are designed for multi-step behaviors, and relative to earlier examples of genetic
engineering, their design is necessarily complex.
How cells can be programmed for such functions
is neither intuitive nor obvious. Here, synthetic
biology has made a radical departure from previous forms of genetic engineering by borrowing
engineering concepts from control theory and
digital computing as a framework upon which to
design genetic circuits for programming cellular
behaviors. A genetic implementation of one such
“simple” computational operation, the Boolean
Figure 1. Cellular computation.
A. Molecular Diagram of a Biological Circuit
Inputs
Arabinose
PBAD
AraC
SicA
Plac
Output
PsicA
sicA
GFP
GFP
InvF
invF
Fluorescence
LacI
IPTG
B. Schematic of an “AND” Gate
Inputs
Arabinose
IPTG
Output
GFP
Inputs
Arabinose
–
+
–
+
Output
IPTG
–
–
+
+
GFP
–
–
–
+
C. Higher-order Circuit
Input 1
Input 2
Output
Input 3
Input 4
Synthetic biology draws some of its inspiration from the engineering disciplines of control theory and digital circuit-design. In
the illustrated example (A), an assemblage of biological components ideally functions to convert two chemical inputs (IPTG
and arabinose) into an output: the expression of the fluorescent reporter protein GFP. Two promoters (PBAD and Plac are each
constitutively repressed until induced by their cognate chemical signals (arabinose and IPTG, respectively). Each operon
expresses half of a two-part transcriptional activator (the SicA and InvF gene products) which together activate the transcription
of the GFP under the control of PsicA. Expression of the reporter only occurs in the presence of both inputs. The DNA circuit can be
represented abstractly as a logic gate implementing the Boolean “AND” operation and is shown with the associated truth table (B).
Higher order circuits (C) can be created by combinations of modular genetic gates; in this example, three AND gates convert four
inputs into a single output. Figure content adapted from Brophy and Voigt (2014).
AND gate, is shown in Figure 1. Higher order
combinations of multiple kinds of genetically
encoded Boolean operations, and other types of
synthetic gene circuits, have been constructed to
perform a variety of simple computational tasks,
including edge-detection, cell to cell communication, and counting of signal inputs (4).
Going from a circuit schematic to a working
genetic device is guided by an engineering paradigm: the design-build-test cycle (Figure 2, p. 4).
A key tool in this process is computer-aided mathematical modeling. Unlike their electrical counterparts, genetic circuits operate under conditions
that dominate the cellular environment. A model
attempting to describe and predict the behavior
of a genetic device must accurately incorporate
a range of parameters such as diffusion, binding
equilibria, networks of protein/DNA interactions,
and dynamic reactant concentrations; a variety of
deterministic and stochastic approaches have been
employed to accomplish this goal (5). As such, the
3
feature article
model embodies a sophisticated hypothesis about
how the device might work. The genetic device
is prototyped (e.g., synthetic DNA is assembled
and transformed into the cell) and its behavior
evaluated in terms of the model. What is learned
during each stage is used to improve the performance of the device in subsequent rounds of the
cycle – through changes to the device itself, as
well as through refinements to the model.
While synthetic biology shares many of the tools
and reagents with hypothesis-driven experimental
biology and molecular biology, it follows a fundamentally different approach. Many of the techniques in a molecular biologist’s repository (e.g.,
oligo synthesis, genome editing) may not exist in
their current form were it not for synthetic biology. Conversely, synthetic biologists can build
upon discoveries made by molecular biologists.
In essence, synthetic biologists assemble genetic components in order to execute an “artificial” function, and in
the process of getting it to work, the engineered genetic
construct becomes itself an object of study and yields
basic principles for application to subsequent designs.
Chemical engineering in vivo
A practical definition of synthetic biology must
also include the latest developments in industrial
fermentation and metabolic engineering. Even a
cursory survey of papers and journals covering
synthetic biology shows a significant number
of reports describing synthetic biology to synthesize fuels, chemicals and materials. Historically, this technology began as an outgrowth of
beer and wine making, after it was discovered
that fermentation could also be used to produce
economically valuable solvents and organic
acids (6). With the advent of greatly expanded
sequence databases, inexpensive DNA synthesis,
and genome engineering methods, it has become
increasingly practical to do chemical synthesis
in vivo. Designer metabolic pathways utilizing
genes encoding enzymes derived from any of the
domains of life can inserted into microbes such as
Saccharomyces cerevisiae or E. coli, endowing them
Figure 2.
Workflow only an engineer could love.
Quantitative
Design
HypothesisDriven
Debugging
Physical
Construction
Experimental
Measurement
The synthetic biology workflow is iterative.
4
with the ability to convert cheap chemical inputs,
such as starch- or cellulose-derived sugars, into
more commercially valuable chemicals.
The products of synthetic biology applied to
industrial fermentation are already in the marketplace and new products are in the works. Large
agro-chemical companies such as Cargill® have
established plants for the conversion of starch to
platform chemicals such as 3-hydroxypropanoate,
which can serve as an intermediate for many other
commodity chemicals. The engineering of E. coli
and Saccharomyces to produce the anti-malarial precursor artemisinic acid by Amyris® is a landmark
achievement in synthetic biology and metabolic
engineering. Elements of their engineered biosynthetic pathway have subsequently been repurposed
to produce fuels and high value chemicals (7),
while Ginkgo Bioworks™ (8) and companies such
as Joule® (9) are engineering microbes to mitigate
greenhouse gases such as methane and CO2 and
produce valuable products, including biofuels. Industrial biosynthesis is not limited to pharmaceuticals and commodity-scale chemicals. Companies
such as Evolva® are working on ways to engineer
yeast to produce vanillin, stevia and even the flavor components of saffron (10), while Pronutria®
is working to efficiently convert CO2 to feed and
medicinal nutrients (11).
The First Synthetic Gene Circuits
In 2000, the first synthetic circuits were made
when Gardner, Cantor and Collins created a genetic toggle switch (12), and Elowitz and Leibler
engineered a repressilator, a synthetic genetic regulatory network designed from scratch to produce
stable oscillations of gene expression (13). Both
of these circuits were model-based, but both also
needed experimental fine-tuning to achieve agreement between model and experimental output.
These experiments were quickly followed by “The
First International Meeting on Synthetic Biology” or SB1.0, which was held in 2004 at MIT
(14). Attended by biologists, chemists, physicists,
engineers and computer scientists, the goal of this
conference was to bring together those scientists
interested in creating and characterizing synthetic
biological systems. This meeting, and smaller ones
like it, laid down the foundation of a new, emerging discipline.
To combat these issues, a public repository, the
Registry of Standard Biological Parts (RSBP), was
founded at MIT by Tom Knight and Drew Endy.
The goal of this repository is to catalog and develop genetic parts into ‘BioBricks®’ that could be
used for the assembly of larger circuits. The BioBrick standard was developed to ensure that parts
could be easily shared and used among synthetic
biologists by requiring submitted parts to conform
to a simplified cloning scheme utilizing four restriction enzymes. However, it became quickly clear
that the task of populating the Registry with biological parts, and the work of characterizing them
to establish their utility, would dwarf the resources
of the relatively small numbers of labs devoted to
synthetic biology. Out of this daunting mission,
and the need to sustainably train a new generation
of synthetic biologists, the International Genetically
Engineered Machine (iGEM) competition was born
(15).
Training the next generation of
bioengineers
Since its inception in 2004, iGEM has evolved into
a highly successful vehicle for training and showcasing a new generation of biological engineers
using the synthetic biology framework. In 2014,
iGEM hosted its 10th annual Jamboree, with over
4,000 participants from across the globe presenting projects that detailed their efforts to model,
build and test genetic devices. Students competed
in a variety of tracks such as Food, Medicine,
Manufacturing and Information Processing. Each
team was also asked to demonstrate that they have
considered the impact and implications of the
technologies they are developing through dialog
with relevant stakeholders. Teams were supported
by various organizations, including NEB (for more
information, visit www.neb.com/igem). To date,
more than 28,000 student competitors have participated in this engineering competition.
A rapidly maturing field
Synthetic Biology as a discipline continues to
grow rapidly. Recent synthetic biology developments include:
A Community to Build From
Circuits Get Complex – In the early 2000s, DNA
circuits continued to advance. More elements were
added (16), and sensing became more diversified
(17,18). Additionally, RNA, not just DNA, was
used in circuit generation (19–21).
As the synthetic biology discipline grew, it quickly
became clear that there needed to be a more efficient way to assemble genetic parts and circuits
(4). Without established methods for assembly
and testing, researchers were forced to ad hoc
experimental designs, wasting time and money by
designing, testing and redesigning constructs.
‘Synthetic’ Used to Investigate ‘Native’ – Beginning in 2009, designed circuits were used to understand native systems through compare/contrast
schemes (22) of engineered versus native systems.
Synthetic Biology was not just limited to engineering new biology; it was also used to investigate and
understand native biology.
www.neb.com
Commercially Valuable Products are Made –
In the 2000s, amino acid biosynthesis was used to
produce commercially valuable products such as
isobutanol (23,24), biodiesel (25) and gasoline (26).
These experiments were a logical extension of fermentation biotechnology and highlighted synthetic
biology’s commercial and environmental promise.
Therapies Engineered – In 2010, Fussenegger
and colleagues engineered a synthetic circuit that,
when inserted into the genome of a mouse mutant
bred to develop hyperuricemia, was able to maintain uric acid homeostasis, essentially correcting an
inborn metabolic defect (33). This demonstrated
the therapeutic promise of synthetic biology.
Assembly of a Whole Bacterial Genome in Yeast
– In 2008, researchers were able to take advantage
of yeast’s remarkable ability to recombine overlapping DNA fragments to assemble an entire genome
in a single step. This method allowed for speedier
assembly of DNA molecules than previous methods
(27,28).
Ongoing Challenges
Synthetic Biology is a young field, but it has
achieved much in a short time period. However,
like all disciplines, it continues to face challenges.
Measurement, Robustness and Predictability
– Aspects of synthetic biology still remain an art.
Genetic circuits often require much “tweaking”
New Genome Editing Tools Emerge – Beginning
in order to get them to function in the context
in 2010, zinc finger nucleases gave way to more
for which they were designed. Further principles
precise genome editing tools, from TALENS (29) to,
governing the function of genetic circuits will have
in 2013, CRISPR/Cas9 (30) systems. This empowto be elucidated to improve the interoperability of
ered synthetic and molecular biologists to create and
genetic parts in multiple contexts.
explore as never before. In addition, a catalytically
inactive form of Cas9, known as dCas9, has further Cells are Not Exactly Digital – Though incredibly powerful as a guiding framework for designenhanced the usefulness of the CRISPR/Cas9 sysing, building, and testing genetic circuits, the digitem by enabling both activation and repression of
transcription in yeast and mammalian cells, allowing tal circuit metaphor has limits. Biological systems
differ from electronic ones in fundamental ways,
modulation of endogenous gene expression (31).
and modeling genetic regulation remains under
First “Artificial Cell” Engineered – In 2010, Craig
determined. Synthetic biology researchers continue
Venter and colleagues demonstrated just how far
to incorporate new ideas and theories to describe,
the discipline of synthetic biology had come when
model and predict genetic circuit behaviors. A new
they published a paper disclosing the recreation of a
and promising area utilizes analogies to analog
Mycoplasma mycoides cell controlled by a chemicallycircuitry (34).
synthesized genome (32).
Ethics and Safety – Synthetic Biology, and indeed
all genetic engineering, has provoked concern over
potential misuse, intentional or accidental. There
is active discussion regarding potential impacts
(35). Built-in forms of biological containment are
also an active area of investigation, including the
refinement of genetic “kill switches”, which ideally would ensure that genetic devices could not
survive outside of the laboratory or factory. Government policy has and will continue to weigh in:
information on the ethics of synthetic biology can
be found in the 2010 Presidential Bioethics Commission report on synthetic biology (36). As with
other technologies, a scientifically literate public is
a requirement for nuanced and effective dialog.
Future directions
The past sixty years have seen incredible scientific
and technological advances based on the ability
to compose in DNA. DNA-driven technologies
will continue to absorb developments and ways of
thinking from diverse fields. Advances in materials
sciences, nanotechnology, microfluidics, automated
liquid handling, indeed all the applied sciences,
will drive new applications using cellular systems
and even biological technologies beyond the cell
(37,38). The proliferation and use of these technologies will continue to impact our lives. With
prudence and foresight, they may prove indispensible to our survival.
See page 8 for references associated with this article.
On the Shoulders of Giants
Bioinformatics
Sequencing Data
Cost of DNA Synthesis
Helping to Establish the Field of Synthetic Biology
Collaboration of biologists
and computer scientists led to
development of software tools for
biologists, including Basic Local
Alignment Search Tool (BLAST),
as well as other alignment and
codon optimization tools (4).
Automated DNA sequencers
became widespread and
more affordable, and the
first complete genome
of an organism was
sequenced (47–49).
Cost of synthesizing DNA dropped
1,000-fold in a decade, and speed
of production increased (50).
Custom Oligonucleotides
Recombinant DNA
In the late 1950s, Khorana
developed the synthetic approach
of blocking/deblocking cycles for
the stepwise elongation of oligos.
This eventually led to solid phase
synthesis and automation.
In 1972, the first published
report of recombinant DNA
made in vitro, and then
transformed into E. coli (42,43).
2010s
1990s
1970s
1950s
2000s
1980s
1960s
Gene Regulation
Polymerase chain
reaction (PCR)
Jacob & Monod first described a genetic
circuit (39), earning a Nobel prize in
1965. Seminal discoveries included the
lysis vs. lysogeny developmental switch
in bacteriophage lambda (λ) (40,41).
First uses of PCR in 1983
(44) helped fuel an explosion
of scientific applications,
from genetic engineering
to forensic science.
Early DNA Assembly
In vitro enzymatic approaches
using complementary,
overlapping oligonucleotides
enabled genes to be synthesized
directly from sequence (45,46).
Genome Editing
One-Step DNA Assembly
First reports connecting
synthetic DNA to genome
editing in the lambda red
system in eubacteria. Later
developments include the
use of engineered zinc finger
nucleases, TALENS (29) and
CRISPR/Cas9 (30,31).
Development of one-step
assembly methods,
including Golden Gate (51),
USER®, Gibson Assembly®
(52) and NEBuilder HiFi
DNA Assembly (53).
5
Which was your first NEB catalog?
– The 40th anniversary prize draw
Which was your first NEB
catalog? Answers from Europe:
With the latest Edition of the NEB Expressions, we asked you “Which was your first NEB catalog?
Simply share your answer with us online and take part in our great 40th anniversary prize draw!”
Here are the results:
We invite you to take a look at the info graphics on the right as well as the nice anecdotes and
comments below you shared with us. Please also visit our webpage at www.NEBanniversary.eu and
find further anecdotes as well the list and photos of the main prize winners.
2013-2014
2011-2013 2009-2010
2002-2003
2000-2001
7,8 % 6,5 % 6,2 %
1998-1999 1996-1997
3,9 % 3,1 %
1992
1990-1991
1988-1989 1986-1987
1985-1986
1982-1983
1981-1982
1980-1981
1977-1978 1975 – 1976
11%
9,9 % 8,0 %
2007-2008
2005-2006
1995
1993-1994
7,8 % 10,9 %
Your comments – a nice selection:
• Instead of handing over a protocol, my
supervisor gave me the NEB catalog when
I took my first steps (...) almost 15 years
ago. It has been my cloning bible ever
since.
• I still keep and work with this catalog. It is
currently on my bench.
• My Professor told me to order my own copy
of the NEB catalog for reference because she
didn‘t loan her out..
• My supervisor was a very nice good-looking
junior postdoc that tried to invite for a beer.
She declined my offer. I had very little experience in Molecular Biolog and she made
me cut DNA. Used to Biochemistry, I used
undiluted 10x buffer for the reaction. She
laughed at me. Next day after she corrected
my experiment. I found a brand-new NEB
catalog on my motorbike with a note: Start
from the last pages, read 2 chapters a day,
learn fast and we‘ll see. The next days I
cleaned my ignorance with joy, laughs and
finally a beer....
• I was announced „most lazy phD-student
of the lab“ as I always try to avoid tedious
steps in my protocol, such as buffer exchange steps. That is why I always use the
NEB-buffer system to perform my enzymatic
reactions.
• My boss told me that the NEB catalog was
the Bible of the lab and that it will bring me
luck! I am always using it right now (the old
one from 1996-97)!
• Often ridiculous stuff happened to me like
when I made my samples and wanted to
load them on the gel, but I did not put a
comb in. Funny, I was looking and looking but could not figure out what did I do
wrong?! I should emphasize that this was
when I got back from my maternity leave,
so my brain was washed out with dirty diapers...:)
• In the first week of my PhD I went to the
lab on Sunday evening at 7.25 pm to do the
inoculum of E. coli. No one told me that the
building closes at 7.30 pm so I spent a nice
Sunday night in the lab. From that moment
on, I always check opening time of the buildings I work in.
8,8 %
2,9 %
0,1 %
4,5 % 1,9 % 1,5 %
0,8 % 1,1 %
1979
1,6 %
0,3 % 0,2 %
1983-1984
1,1 %
0,6 %
Please visit our anniversary webpage
to view the winners and see more nice
and funny comments:
www.NEBanniversary.eu
Order your copy of the
upcoming NEB catalog
2015/16 at your local
distributor!
The lucky winners:
Voucher for free NEB products of choice (value of 1000 €):
5x Quick Cloning Box-HF, essential enzymes & reagents (value of 399 €):
Yukiko Shimada, Friedrich Miescher Institute Basel, Switzerland
Zeljko Jaksic, Ruder Boscovic Institute, Croatia;
Kashif Rasheed, University of Oslo, Norway;
Luka Kranjc, University of Primorska, Slovenia;
Jonas Schaefer, University of Zurich, Switzerland;
Ellen de Waal, Leiden University, The Netherlands
3x Scientific Conference travel grants (value of 500 €):
Valentina André, University of Milano, Italy;
Jeroen Adema, GenDx Utrecht, The Netherlands;
Maria Ángeles Martinez Rodriguez, University of Valencia, Spain
Main Prize
Main Prize
Yukiko Shimada, FMI Basel
Valentina André,
University of Milano
NEB Products of choice
value of 1000 €
Scientific Conference Travel Grant
value of 500 €
To be redeemed at your local NEB distributor
(by Dec. 31th 2015)
40th
anniversary
of NEB
Frankfurt, February 2015, Dr. Thomas Möllenkamp (NEB)
To be redeemed at your local NEB distributor
(by Dec. 31th 2015)
Main Prize
Main Prize
Jeroen Adema,
GenDx, Utrecht
40th
anniversary
of NEB
Frankfurt, February 2015, Dr. Thomas Möllenkamp (NEB)
Scientific Conference Travel Grant
value of 500 €
To be redeemed at your local NEB distributor
(by Dec. 31th 2015)
Maria Ángeles Martinez Rodriguez,
Univ. of Valencia
40th
anniversary
of NEB
Frankfurt, February 2015, Dr. Thomas Möllenkamp (NEB)
Scientific Conference Travel Grant
value of 500 €
To be redeemed at your local NEB distributor
(by Dec. 31th 2015)
40th
anniversary
of NEB
Frankfurt, February 2015, Dr. Thomas Möllenkamp (NEB)
All other lucky winners have been informed and already received their
respective prizes (i.e. 20 NEB Laptop bags and 100 NEB lab timers).
www.neb.com
technical tips
15 Inspiring Scientists Receive
Passion in Science Awards
™
THE 2014 “PASSION IN SCIENCE” AWARDS, hosted by New England Biolabs in celebration
of the company’s 40th anniversary, recognized scientists for their inspirational work that crosses
into the arts, humanitarian service, environmental stewardship and scientific leadership. Selected
from more than 600 candidates worldwide, the Passion in Science awardees provide stirring
examples of the impact scientists can make when choosing to help others.
In October 2014, the awardees gathered from around the world for a two-day summit at NEB’s
campus in Ipswich, Massachusetts, to discuss how scientists can create opportunities to progress
their passions in this first-of-its-kind event.
A working session of the arts and creativity award recipients
includes medical student Shelly Xie, whose evocative sandart performances depict the heartbreaking stories of people
suffering from tropical diseases neglected by modern medicine.
Award recipient Whitney Hagins tours NEB’s greenhouse and
wastewater treatment system with fellow scientific leadership
winners. Hagins, a BioTeach mentor and program coordinator
in Boston, develops hands-on science curriculum and teacher
training to support high school science education.
2014 Passion in Science Award winners pictured with their hosts outside the New England Biolabs facility in Ipswich, MA.
Inspiration in Science Award Winners
Laurie Doering – McMaster University
Jason Furrer – University of Missouri
Whitney Hagins – Massachusetts Biotechnology Education
Ite Laird-Offringa – University of Southern California
Kalai Mathee – Florida International University
Environmental Stewardship Award Winners
Tonni Kurniawan – Xiamen University
Andrew Markley – University of Wisconsin
Humanitarian Duty Award Winners
Lori Baker – Baylor University
Karl Booksh – University of Delaware
Peter Hotez – Sabin Vaccine Institute
Paul McDonald – Virginia Tech Carilion Research Institute
Arts and Creativity Award Winners
Tal Danino – Massachusetts Institute of Technology
Louise Hughes – Oxford Brookes University
Alia Qatarneh – Harvard University
Shelly Xie – UT Southwestern Medical Center
Postdoc Andrew Markley, pictured here in blue, walks the
NEB campus and discusses opportunities to reduce lab waste.
Markley founded an initiative to collect expanded polystyrene
boxes on his University of Wisconsin, Madison campus and
reuse them locally.
Learn more about our award winners and their inspiring projects
in our latest video at www.neb.com/PassionInScience
7
new products
Golden Gate Assembly
advantages of
golden gate assembly
The efficient and seamless assembly of DNA fragments, commonly referred to as 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 only the sequential (3) or simultaneous (4) activities
of a single type IIS restriction enzyme and T4 DNA ligase. This method can be accomplished using
Type IIS restriction enzymes, such as BsaI, and can also be used for the cloning of single inserts. The
assembled fragments, or inserts, can either be precloned or in the amplicon form, where the Type IIs
recognition site is introduced through primer design and PCR. The overhang sequence is not dictated
by the restriction enzyme, and allows the design of appropriate four-base overhang sequences that
lead to scarless assembly. The method is efficient and can be completed in one tube in as little as 5
minutes for single inserts, or can utilize cycling steps for multiple inserts. Golden Gate Assembly has
been widely used in the construction of custom-specific TALENs for in vivo gene editing, among other
applications.
• S eamless cloning – no scar remains
following assembly
• C
an be used to assemble areas of repeats
• C
ompatible with a broad range of fragment
sizes (< 100 bp to > 15 kb)
• E fficient with regions with high GC content
Golden Gate Workflow.
BsaI
3´
BsaI
NNNNNGAGACC
NNNNNCTCTGG 5´
5´ GGTCTCNNNNN 3´
CCAGAGNNNNN
P2
P3
A
P1
B
P5
P4
P6
References:
1. Engler, C, et al. (2008) PLoS ONE, 3: e3647.
2. Engler, C, et al. (2009) PLoS ONE, 4: e5553.
3. Lee, J.H. et al, (1996) Genetic Analysis, 13, 139–145.
4. Padgett, K.A. and Sorge, J.A. (1996) Gene, 168, 31–35.
PCR amplification of
vector and fragments
A
Single-tube reaction
• BsaI
• DNA ligase
PCRlinearized
vector
+
In its simplest form, Golden
Gate Assembly requires a BsaI
recognition site (GGTCTC)
added to both ends of a dsDNA
fragment distal to the cleavage
site, such that the BsaI site is
eliminated in the final product.
B
PCR-amplified
fragments
New England Biolabs supplies reagents for use in Golden Gate Assembly, including restriction
enzymes and ligases. Our new NEB Golden Gate Assembly Mix utilizes two simultaneous enzymatic
activities in a single reaction, specifically digestion with BsaI and ligation with T4 DNA Ligase.
PRODUCT
NEB #
SIZE
NEW NEB Golden Gate Assembly Mix
E1600S
15 reactions
BsaI
R0535S/L
1,000/5,000 units
BsaI-HF
R3535S/L
1,000/5,000 units
BbsI
R0539S/L
300/1,500 units
BsmBI
R0580S/L
200/1,000 units
T4 DNA Ligase
M0202S/T/L/M 2,000/100,000 units
References from feature article (pages 3–5):
1.Jackson, D.A., et al. (1972) Proc. Natl. Acad. Sci. USA., 69:
2904–2909.
2.Morrow, J.F., et al. (1974) Proc. Natl. Acad. Sci. USA.,
71:1743–1747.
3.Elowitz, M., and Lim, W.A. (2010) Nature 468, 889–890.
4.Cameron, D.E., Bashor, C.J. and Collins, J.J. (2014) Nat. Rev.
Microbiol. 12, 381–390.
5.Zheng, Y., et al. (2010) BioMed Research International 2010.
6.Bud, R. (1994) Cambridge University Press.
7.https://amyris.com
8.http://ginkgobioworks.com
9.http://www.jouleunlimited.com/
10.www.evolva.com
11.http://www.pronutria.com/
12.Gardner, T.S., et al. (2000) Nature, 403, 339–342.
8
13.Jones, D.T., et al. (1986) Microbiol. Rev., 50, 484–524.
14.The First International Meeting on Synthetic Biology.
Massachusetts Institute of Technology, Cambridge, MA. June
10-12, 2004. URL: http://syntheticbiology.org/Synthetic_
Biology_1.0.html
15.http://igem.org.
16.Tamsir, A., et al. (2011) Nature, 469, 212–215.
17.Tabor, J.J., et al. (2009). Cell, 137, 1272–1281.
18.Liu, C., et al. (2011) Science, 334, 238–241.
19.Win, M.N., et al. (2008) Science, 322, 456–460.
20.Carothers, J.M., et al. (2011) Science, 334, 1716–1719.
21.Na, D., et al. (2013) Nat. Biotechnol., 31, 170–174 (2013).
22.Cagatay, T., et al. (2009). Cell, 139, 512–522.
23.Atsumi, S., et al. (2008) Nature, 451, 86–89.
24.Huo, Y.X., et al. (2011) Nat. Biotechnol., 29, 346–351.
25.Steen, E.J., et al. (2010) Nature, 463, 559–562.
26.Choi, Y.J., et al. (2013) Nature, 502, 571–574.
27.Gibson, D.G., et al. (2008) Science, 319, 1215–1220.
28.Gibson, D.G., et al. (2008) Proc. Natl. Acad. Sci. USA., 105,
20404–20409.
29.Christian, M., et al. (2010) Genetics, 186, 757–761.
30.Cong, L., et al. (2013) Science, 339, 819–823.
31.Gilbert L.A., et al. (2013) Cell, 154, 442–451.
32.Gibson, D.G., et al. (2010) Science, 329, 52–56.
33.Kemmer, C., et al. (2010). Nat. Biotechnol., 28, 355–360.
34.Sarpeshkar, R. (2014) Philosophical Transactions of the Royal
Society A: Mathematical, Physical and Engineering Sciences
372.2012.
35.Giese, B., and von Gleich, A. (2015) Synthetic Biology. 173–195.
36.http://bioethics.gov/synthetic-biology-report
37.Green A.A., et al. (2014) Cell, 159, 925–939.
38.Pardee. K., et al. (2014) Cell, 159, 940–954.
39.Monod, J., et al. (1961) Cold Spring Harb. Symp. Quant. Biol., 26,
389–401.
40.Ptashne, M. (1986) Cold Spring Harbor Press.
41.Hershey, A., ed. (1971) Cold Spring Harbor Laboratory.
42.Mandel, M., et al. (1970) J. Mol. Biol., 53, 159–162.
43.Cohen, S.N., et al. (1972) Proc. Natl. Acad. Sci. USA., 69,
2110–2114.
44.Mullis, K., et al. (1986) Cold Spring Harb. Symp. Quant. Biol., 51,
263–273.
45.Grundstrom, T., et al. (1985) Nucl. Acids Res. 13, 3305–3316.
46.Dillon, P.J. & Rosen, C.A. (1990) BioTechniques 9, 298–300.
47.Goffeau, A., et al. (1996) Science, 274, 563–567.
48.Blattner, F.R., et al. (1997) Science, 277, 1453–1462.
49.Carlson, R.H. (2011) Harvard University Press.
50. Carlson, R. (2009) Nat. Biotechnol., 27, 1091–1094.
51.Engler, C., et al. (2008) PLOS One, 3, e3647.
52.Gibson, D.G., et al. (2009) Nat. Methods, 6, 343–345.
53.www.NEBuilderHiFi.neb.com
www.neb.com
NEBuilder HiFi DNA Assembly
advantages of nebuilder hifi
NEBuilder HiFi DNA Assembly enables virtually error-free joining of DNA fragments, even those
with 5´- and 3´-end mismatches. Available with and without competent E. coli, this flexible kit
enables simple and fast Seamless Cloning utilizing a new proprietary high-fidelity polymerase.
Find out why NEBuilder HiFi is the next generation of DNA assembly and cloning, supporting the
advancement of both molecular and synthetic biology.
+
Linear vector
• A dapts easily for multiple DNA manipulations,
including site-directed mutagenesis
B
A
DNA inserts with 15-20 bp
overlapping ends (PCR-amplified)
benefits of nebuilder hifi over
gibson assembly master mix
NEBuilder Assembly
A
Single-tube reaction
• NEB Builder Assembly Master Mix:
– Exonuclease chews back
5´ ends to create singlestranded 3´ overhangs
– propriatery DNA polymerase
fills in gaps within each
annealed fragment
• E njoy less screening/re-sequencing of constructs, with virtually error-free, high-fidelity
assembly
B
Assembled
DNA
• J oin DNA fragments together more efficiently,
even with larger fragments or low DNA inputs
Incubate at 50°C
for 15-60 minutes
– DNA ligase seals nicks
in the assembled DNA
• U
se NEBuilder HiFi in successive rounds
of assembly, as it removes 5´- and 3´-end
mismatches
Transformation
DNA Analysis
NEBuilder
HiFi DNA Assembly
Master MixORoffers improved fidelity over Gibson Assembly.
OR
RE Digest
100
10/10 10/10
NEBuilder HiFi DNA Assembly Master Mix
Sequencing
Colony PCR
3/3
5/5
3/3
Gibson Assembly Master Mix
5/5
10/10
Percent Correct Assemblies
80
2/3
40
• B ridge two dsDNA fragments with a synthetic
ssDNA oligo for simple and fast construction
(e.g., linker insertion or gDNA libraries)
• S witch from other systems easily, as
NEBuilder HiFi is compatible with Gibson
Assembly-designed (and other) fragments
• N
o licensing fee requirements from NEB for
NEBuilder products
12/12
11/12
60
• U
se one system for both “standard-size” cloning and large gene assembly products (up to
6 fragments)
• D
NA can be used immediately for transformation, or as a template for PCR or RCA
NEBuilder HiFi DNA Assembly Workflow.
DNA Preparation
• S ave time with simple and fast seamless
cloning
7/10
2/3
5/8
2/5
1/4
20
1/10
0
Fragment size
Vector size
Total size (kb)
Total fragments
assembled
A
B
C
D
E
F
G
H
3 kb
210 bp
210 bp
210 bp
210 bp
3 kb
450 bp + 250 bp
5 x 1 kb frags.
5.4 kb
7.7 kb
7.7 kb
7.7 kb
7.7 kb
2.1 kb
7 kb
3.3 kb
8.4
7.9
7.9
7.9
7.9
5.5
7.7
8.3
2
2
2
2
2
2
3
6
Fidelity of assembled products was compared between NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621) and Gibson
Assembly Master Mix (NEB #E2611). Experiments were performed using various fragment and vector sizes following suggested
protocols. Experiments B through E varied because sequences of fragments were different. Experiments F and H were
performed with fragments containing 3´ end mismatches.
PRODUCT
NEB #
SIZE
NEBuilder HiFi DNA Assembly Master Mix
E2621S/L/X
10/50/250 rxns
NEBuilder HiFi DNA Assembly Cloning Kit
(includes competent cells)
E5520S
10 rxns
Visit NEBuilderHiFi.com
to learn more!
Request your FREE Sample*
from your local distributor!
*as long as supplies last!
9
Purple is the new Black...
Quick-Load Purple DNA Ladders
1
2
Lane 1. Invitrogen™ TrackIt™
1 Kb Plus DNA Ladder; Lane
2. NEB Quick-Load Purple
2-log DNA Ladder; 1 µg per
gel lane
PRODUCT
Quick-load Purple 2-Log DNA Ladder
Quick-load Purple 100 kb DNA Ladder
Quick-load Purple 1kb DNA Ladder
kb
10.0
8.0
6.0
5.0
4.0
3.0
ng
40
40
48
40
32
120
2.0
40
1.5
57
1.2
45
1.0
0.9
0.8
122
34
31
0.7
27
0.6
23
0.5
124
0.4
49
0.3
37
0.2
32
0.1
61
• Quickly
determine the size of your DNA, using
evenly-spaced bands and easily-identifiable
reference bands
kb
ng
10.0 42
8.0 42
6.0
5.0
50
42
4.0
33
3.0
125
2.0
48
• Improve
your ability to discriminate between
bands, with our new & improved purple dye
bp
1,517
ng
45
1,200
35
1,000
900
95
27
800
24
700
1.5
600
36
500/517
18
97
Please request
your free NEB
DNA Ladder
magnet from your
local distributor!
DNA Ladders - the Bestsellers
Low Range and Fast DNA Ladders
kb
ng
10.0 40
8.0 40
6.0 48
5.0 40
4.0 32
3.0 120
kb ng
10.0 42
8.0 42
6.0
50
5.0
42
4.0
33
3.0
125
2.0
48
1.5
36
1.0
42
0.5
42
kb
40
20
15
bp
ng
1,350 103
10
916
70
8
766
700
650
600
550
500
450
58
54
50
46
42
76
34
500 27
400
31
250 27
350
27
300
46
200 110
0.766 43
250
57
200
107
150
46
100
69
bp
1,517
ng
45
2.0
40
1,200
35
1.5
57
4
1,000
95
900
27
1.2
45
3
800
24
700
21
1.0
0.9
0.8
122
34
31
2
600
18
0.7
27
500/517
97
0.6
23
0.5
124
400
38
0.4
49
0.3
37
0.2
32
0.1
61
300
29
200
25
100
48
6
5
bp ng
766 42
350 20
150 33
100 43
1.5
1
75
58
50
63
25
50
0.5
1.0
0.5
400
38
300
29
200
25
100
48
Quick-Load Purple
1 kb DNA Ladder:
Quick-Load Purple
100 bp DNA Ladder:
NEB #N0550
NEB #N0552
NEB #NN0551
Optional, the unique OneTaq Quick-Load
One-Step Reaction Mix included allows for
direct gel loading.
The kit is capable of multiplex detection of two
or three targets.
10
NEB #
SIZE
OneTaq One-Step RT-PCR Kit
E5315 S
30 rxns
61
Quick-Load 1 kb Extend DNA Ladder
1.3% TAE agarose gel.
Mass values are for
0.5 µg/lane.
1.0% TBE agarose gel.
Mass values are for
1 µg/lane.
0.6% TBE agarose gel.
Mass values are for 0.5 µg/lane.
1.8% TBE agarose gel.
Mass values are for
1 µg/lane.
1.8% TBE agarose gel.
Mass values are for 0.5 µg/lane.
1.2% TBE agarose gel.
Mass values are for
0.5 µg/lane.
NEB #N3231
NEB #N3200
NEB #N3236
NEB #N3233
NEB #N3238
Quick-Load
1 kb DNA Ladder:
NEB #N0468
Quick-Load
100 bp DNA Ladder:
NEB #N0467
Quick-Load
2-Log DNA Ladder:
NEB #N0469
• Quick-Load Ladders: contain
bromophenol blue
TriDye
1 kb DNA Ladder:
NEB #N3272
TriDye
100 bp DNA Ladder:
NEB #N3271
TriDye
2-Log DNA Ladder:
NEB #N3270
• TriDye Ladders: contain xylene cyanol
FF, bromophenol blue and orange G
Quick-Load Purple
1 kb DNA Ladder:
NEB #N0552
Quick-Load Purple
100 bp DNA Ladder:
NEB #NN0551
Quick-Load Purple
2-Log DNA Ladder:
NEB #N0550
NEB #N3239S
Order your favorite as:
• Quick-Load Purple Ladders:
contain dyes that cast no UV shadow
for added convenience
and “ready-to-load”!
Quick-Load
50 bp DNA Ladder:
NEB #N0473
Fast DNA Ladder
Quick-Load
Low Molecular Weight
DNA Ladder:
NEB #N0474
Toll Free: (Germany) 0800/246-5227
Toll Free: (Austria) 00800/246-52277
www.neb-online.de
[email protected]
Discount!
Ask your local
distributor for details!
Offer closes
June 30th, 2015!
•
• RT step at 42°C - 55°C
PRODUCT
41
0.05
NEB #N3232
• Detect
at little as 0.1 pg of a GAPDH target
For more information, visit www.international.neb.com/E5315
33
0.15
1 kb DNA Ladder
• Save
time by combining cDNA synthesis and
PCR in a single reaction
About 100 ng of Jurkat total RNA was used in 50 μl reactions
following the standard protocol. The marker lane (M) contains
Quick-Load 2-log DNA Ladder (NEB #N0469).
40
0.3
0.8% TAE agarose gel.
Mass values are for
0.5 µg/lane.
• Sensitive
and robust end-point detection of
RNA templates
Detection of RNA templates of different length.
108
0.5
43
Low Molecular Weight
DNA Ladder
advantages
The kit combines an optimized enzyme mix with
robust reagents for optimal results.
28
1.0
84
OneTaq One-Step RT-PCR Kit –
Faster protocols with less hands-on time!
M 0.7 1.11.92.32.55.57.69.3 kb
38
1.5
50 bp DNA Ladder
Find all Markers and Ladders at http://international.neb.com/products/markers-and-ladders
The OneTaq One-Step RT-PCR Kit offers sensitive and robust end-point detection of RNA
templates. cDNA synthesis and PCR amplification steps are performed in a single reaction using
gene-specific primers, resulting in a streamlined
RT-PCR protocol!
36
2.0
2-Log DNA Ladder
now at Special
SIZE
125-250 gel lanes
125 gel lanes
125 gel lanes
ng
36
36
3.0
100 bp DNA Ladder
42
42
kb
10.0
5.0
300 33
NEB_GmbH_Magnetic_DNA_Ladder_card_15.indd 1
Quick-Load Purple
2-Log DNA Ladder:
NEB #
N0550S
N0551S
N0552S
21
• Easily
view smaller-sized bands, as our purple
dye casts no UV shadow
DNA MARKERS & LADDERS
Nobody likes a UV shadow on their gels!
With NEB’s Quick-Load Purple DNA Ladders,
you will see sharper bands and no UV shadow
at the dye front thanks to our new purple gel
loading dye.
advantages
• Robust
amplification of amplicons
from 100 bp to 9 kb
• Faster
protocols with less hands-on time
• Quick-Load
Reaction Mix allows instant gel
loading
11.02.15 17:09
www.neb.com
Rapid PNGase F –
Complete deglycosylation in minutes
Effective manufacturing of therapeutic proteins requires characterizing their N-glycosylation in the
shortest time possible. Rapid PNGase F is an improved reagent that allows the complete and rapid
deglycosylation of antibodies and immunoglobulin fusion proteins, as well as other glycoproteins.
All N-glycans are released in five minutes without bias, and are ready to be prepared for downstream
chromatography or mass spectrometry analysis. Rapid PNGase F creates an optimized workflow,
reducing processing time without compromising sensitivity or reproducibility.
advantages
• Convenient
one-step reaction compatible
with high throughput applications
• Complete
deglycosylation of antibodies and
immunoglobulin fusion proteins in minutes
• R elease of all N-glycans without bias,
compatible with downstream chromatography
or mass spectrometry analysis
• R ecombinant source
• O
ptimal activity is ensured for 12 months
ESI-TOF analysis of an antibody before (left) and after (right) treatment
with Rapid PNGase F
• Purified
to >99% homogeneity
Get free
Application Note:
“Unbiased and fast
IgG deglycosylation
for accurate N-glycan
analysis using
Rapid PNGase F”
PRODUCT
NEB #
SIZE
Rapid PNGase F
P0710S
50 reactions
Visit www.international.neb.com/P0710
to download the full application note.
Near-infrared Imaging in Living Cells
New England Biolabs now offers a unique cell-permeable near-infrared fluorescent probe that enables
live-cell imaging of intracellular proteins using the SNAP-tag® technology.
SNAP-Cell® 647-SiR (SiR-SNAP) is excited at around 650 nm and emits around 670 nm, has a high
quantum efficiency in aqueous media, and is stable against photobleaching. The excellent spectroscopic properties of SNAP-Cell 647-SiR, combined with its high permeability, make it ideally suited
for super-resolution microscopy of cellular proteins in living cells and in vivo.
Recent Publications using SNAP-tag
Jaensch, N. et al. (2014) Stable cell surface expression of GPI-anchored proteins, but not intracellular
transport, depends on their fatty acid structure. Traffic, 15, 1305-1329.
Sun, X. et al. (2014) Probing homodimer formation of epidermal growth factor receptor by selective
cross linking. Euro. J. Med. Chem. 88, 34-41.
Yang, G. et al. (2014) Genetic targeting of chemical indicators in vivo. Nature Methods, doi: 10.138/
NMETH.3207.
Lukinavicius, G. et al. (2013) A near-infrared fluorophore for live-cell superresolution microscopy of
cellular proteins. Nature Chemistry, 5, 132-139.
PRODUCT
NEB #
SIZE
SNAP-Cell 647-SiR
S9102S
30 nmol
Composite image in confocal (lower left) and super
resolution (upper right) showing live U2-OS cells expressing
a centrosomal fusion protein, Cep41-SNAP, labeled with
SNAP-Cell 647-SiR. Scale bar is 1 μm.
Learn more about the
SNAP-tag technology at
www.neb.com/SNAPtag
11
DNA CLONING
DNA AMPLIFICATION & PCR
EPIGENETICS
RNA ANALYSIS
LIBRARY PREP FOR NEXT GEN SEQUENCING
PROTEIN EXPRESSION & ANALYSIS
CELLULAR ANALYSIS
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938-2723
Your local NEB distributor:
Rapid PCR Cleanup Enzyme Set –
Test it now and get your FREE Sample*!
The NEB Rapid PCR Cleanup Enzyme Set consists of two recombinant enzymes, Exonuclease I
(Exo I) and Shrimp Alkaline Phosphatase (rSAP). It allows for rapid and complete enzymatic
degradation of residual PCR primers and dephosphorylation of dNTPs subsequent to PCR.
The Rapid PCR Cleanup Enzyme Set enables direct downstream sequencing without the need for
additional purification steps. The components are added directly to the PCR reaction after thermal
cycling and are 100% compatible with commonly used PCR reaction buffers.
For more product details, please visit
www.neb-online.eu/e2622
advantages
Primer
PCR product
dNTPs
• Fast 15 minute protocol
• Add directly to PCR product
• 100% Sample Recovery
+ Rapid PCR Cleanup Enzyme Set
(1 µl of each enzyme per 5 µl of
PCR product)
Incubate at 37°C
for 5 minutes
• Scalable for different reaction sizes
• No interference on downstream applications
• Easy to automate
Inactivate at 80°C
for 10 minutes
Cleaned up
PCR product
PCR products contain residual primer and dNTPs that handicap
downstream sequencing. Rapid PCR Cleanup Enzyme Set
degrades excess primers and dNTPs. Add 1 µl of each enzyme
(Exonuclease I and rSAP) to 5 µl of PCR product (scalable) and
incubate for only 5 minutes at 37°C. The reaction is stopped by
enzyme inactivation at 80°C for 10 minutes. The temperature
regime can be performed in a thermocycler for maximum
convenience.
Get your FREE
Sample*
Ask your local
distributor for details!
Offer closes
June 30th, 2015!
* limited offer as long as supplies last.
PRODUCT
NEB #
SIZE
Rapid PCR Cleanup Enzyme Set, sample size
E2622V
20 rxns (free)*
Rapid PCR Cleanup Enzyme Set
E2622S/L
100/500 rxns
Rapid PCR Cleanup Enzyme Set
E2622X/E
2000 / 5000 rxns
BeNeLux:
BIOKÉ
Schuttersveld 2
2316 ZA Leiden
Tel: (+31) 71 720 0220
(BE: 0800 - 71640)
Fax: (+31) 71 891 0019
[email protected]
www.bioke.com
NORWAY:
BioNordikaBergman AS
Gladengveien 3 B
0661 Oslo
Tel +47 23 03 58 00
Fax +47 23 03 58 01
[email protected]
www.bionordikabergman.no
CZECH REPUBLIC:
BIOTECH A.S.
Sluzeb 4
108 52 Praha 10
Tel: (Toll Free) 0800 124683
Fax: 02 72701742
[email protected]
www.biotech.cz
POLAND:
Lab-JOT Ltd. Sp.z o.o. Sp.k.
Al. Jerozolimskie 214,
02-486 Warsaw
stacjonarny: +48 22 335 988 4
komórkowy: +48 606 338 879
Fax: (+48 22) 335 981 9
[email protected]
www.labjot.com
DENMARK:
BioNordika Denmark A/S
Marielundvej 48
2730 Herlev
DENMARK
Tel: (39) 56 20 00
Fax: (39) 56 19 42
[email protected]
www.bionordika.dk
PORTUGAL:
Werfen
Rua do Proletariado
1 - Quinta do Paizinho
2790-138 Carnaxide
Tel. +351 214 247 312
Fax + 351 214 172 674
[email protected]
http://pt.werfen.com
FINLAND:
BioNordika Finland OY
Kutomotie 18
00380 Helsinki
Tel: +358/207/410 270
Fax +358/207/410 277
[email protected]
www.bionordika.fi
SWEDEN:
BIONORDIKA SWEDEN AB
Norrbackagatan 47A
113 34 Stockholm
Tel: (08) 30 60 10
Fax: (08) 30 60 15
[email protected]
www.bionordika.se
GREECE:
BIOLINE SCIENTIFIC
1 Meg. Alexandrou Str.
10437 Athens
Tel: 210 5226547
Fax: 210 5244744
[email protected]
www.bioline.gr
SPAIN:
Werfen Plaza de Europa, nº 21-23
08908 L’Hospitalet de Llobregat
(Barcelona)
Tel: +34 902 20 30 90
Fax: +34 902 22 33 66
[email protected]
http://es.werfen.com
HUNGARY:
KVALITEX KFT
Pannónia u. 5.
1136 Budapest
Phone: (1) 340-4700
Fax: (1) 339-8274
[email protected]
www.kvalitex.hu
ISRAEL:
ORNAT LTD.
POB 2071
Rehovot 76120
Tel: +972-8-9477077
Fax: +972-8-9363934
[email protected]
www.ornat.co.il
ITALY:
EUROCLONE S.P.A.
Via Figino 20/22
20016 Pero (Milan)
Free call: 800-315911
Tel: (02) 381951
Fax: (02) 38101465
[email protected]
www.euroclonegroup.it/celbio/
Printed in Germany
SLOVENIA:
Mikro+Polo d.o.o.
Zagrebška cesta 22
SI-2000 Maribor
Tel: +386 2 614 33 00
Fax: +386 2 614 33 20
[email protected]
www.mikro-polo.Si/
SWITZERLAND:
BIOCONCEPT
Paradiesrain 14
Postfach 427
CH 4123 Allschwil 1
Tel: (061) 486 80 80
Fax: (061) 486 80 00
[email protected]
www.bioconcept.ch
TURKEY:
SACEM HAYAT TEKNOLOJILERI
Gebze Plastikçiler Organize Sanayi
Bölgesi (GEPOSB)
Cumhuriyet Cad. No:3 41400
Gebze/KOCAELI
Tel: 0262 751 02 74
Fax: 0262 751 02 75
[email protected]
www.sacem.com.tr