Special Cloning Edition
NEB EXPRESSIONS
®
A scientific update from New England Biolabs
Foundations of Molecular Cloning –
Past, Present & Future page 3
Top DNA Ligases at Special Price page 9
New Products to Facilitate your Cloning page 10
Essential Reagents – Exceptional Price:
NEB Quick Cloning Box-HF page 13
Spring, 2014
rsaP
advantages
DNAphosphorylationisakeystepinthecloningworkflow.Foryournextcloningexperiment,
considerShrimpAlkalinePhosphatase(rSAP).rSAPisaheatlabilealkalinephosphatasepurified
fromarecombinantsource.rSAPcontainsnoaffinitytagsorothermodifications.
rSAPheatinactivationat65°C
100
Remaining Activity (%)
80
SER™
70
60
Clone with
Confidence
50
40
30
20
10
10
15
5
Incubation at 65°C, minutes
20
Issue III, 2013
• Use less enzyme; very high specific
activity compared to mammalian
20
1 unit of rSAP was incubated under recommended
reaction conditions, including DNA, for 30 minutes,
and then heated at 65°C. Remaining phosphatase
activity was measured by PNPP assay.
Past, Present & Future
Understanding where we’ve come from is essential to knowing where
we’re going. Brush up on the history of molecular cloning, and learn
where it could take us in the future.
0
0
•Reagents
for every
Quick-Load Purple 2-Log
Ladder
201dna
0
step in the workflow
gel Loading dye, Purple (6X)
8 Cloning Reagents & Technical Guide
kb
10.0
8.0
6.0
5.0
4.0
3.0
NEB’spopular2-LogDNALadderisnowavailableinaQuick-Loadformat
2010
of a whole genome
(41,42)
•Educational tools
and technical support
• Convenient, ready-to-load format
2.0
1.5
• tighter, brighter bands with new
dye format
special price
1.2
1.0
0.9
0.8
• Coming soon – free tube of
gel Loading dye, Purple with all
HF restriction enzymes
0.7
0.6
new products
0.5
0.4
now avaiLaBLe
Quick-Load Purple
2-Log dna Ladder
Nowwithpurpledye
0.3
We want you to clone with confidence; that’s why we continuously strive
to develop best-in-class solutions for your molecular cloning needs.
0.2
0.1
2-Log DNA Ladder
1.0% TBE agarose gel.
OrderingInformation
ProdUCt
neB #
NEBEssentialCloningSet
E1400S
1set
Quick-LoadPurple2-LogDNALadder
N0550S
ShrimpAlkalinePhosphatase(rSAP)
B7024S NEB Stable
4ml Competent
E. coli (High Efficiency)
E1202S
20rxns
C3040H
Lot: 134
x 0.05 ml/tube
Store at -80°C
M0371S/LH 20 500/2500units
125–250gellanes
NEBStableCompetentE. coli (HighEfficiency)
C3040H
C3040H 001131114111
12 Cloning Competent Cells
featured products
siZe
CAUTION: This product contains DMSO,
a hazardous material. Review the MSDS before handling.
GelLoadingDye,Purple(6X)
NEBPCRCloningKit
• analyze dna fragments from
0.1–10 kb
9 Top DNA Ligases at Special Price
10 New Cloning Tools to Enhance your Research
genome using Gibson
Assembly® (39)
&
Introduction of
High-Fidelity (HF®)
restriction enzymes
Explore the wise choice at
www.CloneWithNEB.com
advantages
new brochures
fordirectgelloading.ThisproductutilizesournewGelLoadingDye,
First synthetic life –
•Novel solutions
–
Purple,whichresultsinbrighter,sharperbandswhencomparedtoglycerolMycoplasma mycoides
2008
baseddyes.Additionally,thisnewdyeleavesnoUVshadow.Tryit,andsee
(45)
Synthesis of the
howitstacksup.
including2000
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Assembly
mycoplasma genitalium
in vitro synthesis
5
3 Foundations of Molecular Cloning –
phosphatases
featurealkaline
article
20
shares the
Prize for his
n PCR
0
• add directly to restriction enzyme
digests; active in all neBuffers
05
0
2007
Introduction of ligationindependent
cloning (35)
• experience significantly improved
stability, compared to native enzyme
• no need for supplemental additives,
such as zinc
Contents
90
• inactivate rsaP completely, in
5 minutes at 65°C
Scan for
product
info.
®
Learn about the benefits of using NEB Competent E. coli for cloning.
Our selection chart will help you decide which strain is best for you.
20x0.05ml/tube
Genotype: F´ proA+B+ lacIq ∆(lacZ)M15 zzf::Tn10
(TetR)/∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16
galE15 e14- φ80dlacZ∆M15 recA1 relA1 endA1 nupG
rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS-mcrBC)
Transformation Efficiency: 1–5 x 108 cfu/µg
pUC19 DNA
Resistance: T1 phage (fhuA), Strep, Tet
Sensitivity: Amp, Cam, Kan, Nit, Spec
For Research Use Only.
New England Biolabs is an ISO 9001,
ISO 14001 and ISO 13485 certified facility.
11
13 NEB Quick Cloning Box-HF
featured product
The most commonly used cloning enzymes and tools at a price you
can’t afford to miss.
14 Gibson Assembly Cloning Kit
featured product
®
Transform your cloning with NEB’s Gibson Assembly Cloning Kit.
Assemble and clone your insert in just under two hours.
15 Robust Colony PCR from Multiple E. coli Strains
application note
using OneTaq ® Quick-Load® Master Mixes
OneTaq DNA Polymerase has been formulated for robust yields
with minimal optimization. See how it performs in colony PCR with
18 different E. coli strains.
cover photo
Transformed E. coli grow on agar plates in a blue/white screening assay.
HF®, NEW ENGLAND BIOLABS®, NEB®, NEBUILDER®, NEBCUTTER®, ONETAQ®, Q5®, QUICK-LOAD®, REBASE® and SHUFFLE® are registered trademarks
of New England Biolabs, Inc.
CUTSMART™, DEEP VENT™, QUICK BLUNTING™, QUICK LIGATION™ and USER™ are trademarks of New England Biolabs, Inc.
FSC® is a registered trademark of the Forest Stewardship Council. GIBSON ASSEMBLY® is a registered trademark of Synthetic Genomics, Inc.
GENEART® is a registered trademark of Life Technologies, Inc. GATEWAY®, ONESHOT® and MAX EFFICIENCY® are registered trademarks of Invitrogen.
IN-FUSION® is a registered trademark of Clontech Laboratories, Inc. NANOSPEC® is a registered trademark of Thermo Fisher Scientific. DH5α™, DH10B™ and
MACH1™ are trademarks of Invitrogen.
2
www.neb.com
feature article
Foundations of Molecular Cloning – Past, Present & Future
Molecular cloning, a term that has come to mean the creation of recombinant DNA molecules, has spurred progress throughout the life
sciences. Beginning in the 1970s, with the discovery of restriction endonucleases – enzymes that selectively and specifically cut molecules
of DNA – recombinant DNA technology has seen exponential growth in both application and sophistication, yielding increasingly powerful
tools for DNA manipulation. Cloning genes is now so simple and efficient that it has become a standard laboratory technique. This has led
to an explosion in the understanding of gene function in recent decades. Emerging technologies promise even greater possibilities, such as
enabling researchers to seamlessly stitch together multiple DNA fragments and transform the resulting plasmids into bacteria, in under two
hours, or the use of swappable gene cassettes, which can be easily moved between different constructs, to maximize speed and flexibility.
In the near future, molecular cloning will likely see the emergence of a new paradigm, with synthetic biology techniques that will enable
in vitro chemical synthesis of any in silico-specified DNA construct. These advances should enable faster construction and iteration of DNA
clones, accelerating the development of gene therapy vectors, recombinant protein production processes and new vaccines.
Rebecca Tirabassi, Bitesize Bio.
The Foundation of Molecular Cloning
Introduction
Cutting (Digestion). Recombinant DNA
technology first emerged in the late 1960s, with
the discovery of enzymes that could specifically
cut and join double-stranded DNA molecules. In
fact, as early as 1952, two groups independently
observed that bacteria encoded a “restriction
factor” that prevented bacteriophages from
growing within certain hosts (1,2). But the nature
of the factor was not discovered until 1968, when
Arber and Linn succeeded in isolating an enzyme,
termed a restriction factor, that selectively cut
exogenous DNA, but not bacterial DNA (3).
These studies also identified a methylase enzyme
that protected the bacterial DNA from restriction
enzymes.
Molecular cloning refers to the isolation of a DNA
sequence from any species (often a gene), and its
insertion into a vector for propagation, without
alteration of the original DNA sequence. Once
isolated, molecular clones can be used to generate
many copies of the DNA for analysis of the gene
sequence, and/or to express the resulting protein
for the study or utilization of the protein’s function. The clones can also be manipulated and mutated in vitro to alter the expression and function
of the protein.
The basic cloning workflow includes four steps:
1.Isolation of target DNA fragments
(often referred to as inserts)
2.Ligation of inserts into an appropriate cloning
vector, creating recombinant molecules
(e.g., plasmids)
3.Transformation of recombinant plasmids into
bacteria or other suitable host for propagation
4.Screening/selection of hosts containing the
intended recombinant plasmid
These four ground-breaking steps were carefully
pieced together and performed by multiple laboratories, beginning in the late 1960s and early
1970s. A summary of the discoveries that comprise traditional molecular cloning is described in
the following pages.
Shortly after Arber and Linn’s discovery, Smith
extended and confirmed these studies by isolating
a restriction enzyme from Haemophilus influenza.
He demonstrated that the enzyme selectively cut
DNA in the middle of a specific 6 base-pair stretch
of DNA; one characteristic of certain restriction
enzymes is their propensity to cut the DNA
substrate in or near specific, often palindromic,
“recognition” sequences (4).
The full power of restriction enzymes was not realized until restriction enzymes and gel electrophoresis were used to map the Simian Virus 40 (SV40)
genome (5). For these seminal findings, Werner
Arber, Hamilton Smith, and Daniel Nathans shared
the 1978 Nobel Prize in Medicine.
Figure 1. Traditional Cloning Workflow
DNA Preparation
MCS
Fragment A
(PCR-amplified or
annealed oligos)
+
Vector B
MCS
OR
Vector A
+
Restriction
Enzymes
Restriction
Enzymes
Digested
Fragment A
Vector B
+
Ligation
Digested
Vector B
Digested
Vector A
+
Digested
Vector B
DNA ligase
Transformation
Assembled
DNA
Using PCR, restriction sites are added to both ends of a dsDNA, which is then digested by the corresponding restriction enzymes (REases).
The cleaved DNA can then be ligated to a plasmid vector possessing compatible ends. DNA fragments can also be moved from one vector
into another by digesting with REases and ligating with compatible ends of the target vector. Assembled construct can then be transformed
into Escherichia coli (E. coli).
3
feature article continued…
Assembling (Ligation). Much like the discovery
of enzymes that cut DNA, the discovery of an
enzyme that could join DNA was preceded by
earlier, salient observations. In the early 1960s,
two groups discovered that genetic recombination
could occur though the breakage and ligation
of DNA molecules (6,7), closely followed by the
observation that linear bacteriophage DNA is
rapidly converted to covalently closed circles after
infection of the host (8). Just two years later, five
groups independently isolated DNA ligases and
demonstrated their ability to assemble two pieces
of DNA (9-13).
Not long after the discovery of restriction enzymes
and DNA ligases, the first recombinant DNA molecule was made. In 1972, Berg separately cut and
ligated a piece of lambda bacteriophage DNA or
the E. coli galactose operon with SV40 DNA to
create the first recombinant DNA molecules (14).
These studies pioneered the concept that, because
of the universal nature of DNA, DNA from any
species could be joined together. In 1980, Paul
Berg shared the Nobel Prize in Chemistry with
Walter Gilbert and Frederick Sanger (the developers of DNA sequencing), for “his fundamental
studies of the biochemistry of nucleic acids, with
particular regard to recombinant DNA.”
Transformation. Recombinant DNA technology
would be severely limited, and molecular cloning
impossible, without the means to propagate and
isolate the newly constructed DNA molecule. The
ability to transform bacteria, or induce the uptake,
incorporation and expression of foreign genetic
material, was first demonstrated by Griffith when
he transformed a non-lethal strain of bacteria into
a lethal strain by mixing the non-lethal strain with
heat-inactivated lethal bacteria (15). However, the
nature of the “transforming principle” that conveyed lethality was not understood until 1944. In
the same year, Avery, Macleod and McCarty demonstrated that DNA, and not protein, was responsible for inducing the lethal phenotype (16).
Initially, it was believed that the common bacterial
laboratory strain, E. coli, was refractory to transformation, until Mandel and Higa demonstrated that
treatment of E. coli with calcium chloride induced
the uptake of bacteriophage DNA (17). Cohen
applied this principle, in 1972, when he pioneered
the transformation of bacteria with plasmids to
confer antibiotic resistance on the bacteria (18).
The ultimate experiment: digestion, ligation and
transformation of a recombinant DNA molecule
was executed by Boyer, Cohen and Chang in
1973, when they digested the plasmid pSC101
with EcoRI, ligated the linearized fragment to
another enzyme-restricted plasmid and transformed
the resulting recombinant molecule into E. coli, conferring tetracycline resistance on the bacteria (19),
thus laying the foundation for most recombinant
DNA work since.
Building on the Groundwork
While scientists had discovered and applied all of
the basic principles for creating and propagating
recombinant DNA in bacteria, the process was
inefficient. Restriction enzyme preparations were
unreliable due to non-standardized purification
procedures, plasmids for cloning were cumbersome,
difficult to work with and limited in number, and
experiments were limited by the amount of insert
DNA that could be isolated. Research over the next
few decades led to improvements in the techniques
and tools available for molecular cloning.
Early vector design.
Development of the first standardized vector. Scientists
working in Boyer’s lab recognized the need for
a general cloning plasmid, a compact plasmid
with unique restriction sites for cloning in foreign
DNA and the expression of antibiotic resistance
genes for selection of transformed bacteria. In
1977, they described the first vector designed for
cloning purposes, pBR322 (20). This vector was
small, ~4 kilobases in size, and had two antibiotic
resistance genes for selection.
A History of Molecular Cloning
1971
Restriction enzyme
mapping of the
simian virus SV40 (5)
1974
NEB opens
for business
1970
Isolation of
restriction
enzymes that
selectively cut (4)
1961
Genetic recombination
demonstrated (6,7)
1977
Report of the first
cloning vector
(pBR322) (20)
1978
Nobel Prize
awarded to Smith,
Arber and Nathans
for the discovery of
restriction enzymes
5
197
1952 – 53
Genetic demonstration
of phage restriction
(1,2)
1975
Launch of REBASE
(Restriction Enzyme
Database)
80
1950
65
60
19
19
1970
5
195
4
19
1967
Isolation of the
first DNA ligases
(9–13)
1968
Isolation of the first
restriction factor that
could selectively cut
bacteriophage DNA (3)
1976 – 77
Introduction of
Maxam-Gilbert and
Sanger sequencing
(26,27)
1972
Assembly of the first
recombinant DNA
&
Tranformation of the
first E. coli (17,18)
www.neb.com
Vectors with on-board screening and higher yields.
Although antibiotic selection prevented nontransformed bacteria from growing, plasmids that
re-ligated without insert DNA fragments (selfligation) could still confer antibiotic resistance on
bacteria. Therefore, finding the correct bacterial
clones containing the desired recombinant DNA
molecule could be time-consuming.
Vieira and Messing devised a screening tool to
identify bacterial colonies containing plasmids
with DNA inserts. Based upon the pBR322
plasmid, they created the series of pUC plasmids,
which contained a “blue/white screening” system
(21). Placement of a multiple cloning site (MCS)
containing several unique restriction sites within
the LacZ´ gene allowed researchers to screen for
bacterial colonies containing plasmids with the
foreign DNA insert. When bacteria were plated
on the correct media, white colonies contained
plasmids with inserts, while blue colonies
contained plasmids with no inserts. pUC plasmids
had an additional advantage over existing vectors;
they contained a mutation that resulted in higher
copy numbers, therefore increasing plasmid yields.
Improving restriction digests. Early work
with restriction enzymes was hampered by the
purity of the enzyme preparation and a lack of
understanding of the buffer requirements for
each enzyme. In 1975, New England Biolabs
(NEB) became the first company to commercialize
restriction enzymes produced from a recombinant
source. This enabled higher yields, improved
purity, lot-to-lot consistency and lower pricing.
Currently, over 4,000 restriction enzymes,
recognizing over 300 different sequences, have
been discovered by scientists across the globe
[for a complete list of restriction enzymes and
recognition sequences, visit REBASE® at rebase.
neb.com (22)]. NEB currently supplies over 230
of these specificities.
NEB was also one of the first companies to
develop a standardized four-buffer system, and
to characterize all of its enzyme activities in this
buffer system. This led to a better understanding
of how to conduct a double digest, or the
digestion of DNA with two enzymes simultaneously. Later research led to the development
of one-buffer systems, which are compatible with
the most common restriction enzymes (such as
NEB’s CutSmart™ Buffer).
With the advent of commercially available
restriction enzyme libraries with known sequence
specificities, restriction enzymes became a
powerful tool for screening potential recombinant
DNA clones. The “diagnostic digest” was, and still
is, one of the most common techniques used in
molecular cloning.
1991
Introduction of USER™
cloning (36)
Vector and insert preparation. Cloning
efficiency and versatility were also improved
by the development of different techniques for
preparing vectors prior to ligation. Alkaline
phosphatases were isolated that could remove
the 3´ and 5´ phosphate groups from the ends
of DNA [and RNA; (23)]. It was soon discovered
that treatment of vectors with Calf-Intestinal
Phosphatase (CIP) dephosphorylated DNA
ends and prevented self-ligation of the vector,
increasing recovery of plasmids with insert (24).
The CIP enzyme proved difficult to inactivate,
and any residual activity led to dephosphorylation
of insert DNA and inhibition of the ligation
reaction. The discovery of the heat-labile alkaline
phosphatases, such as recombinant Shrimp
Alkaline Phosphatase (rSAP) and Antarctic
Phosphatase (AP) (both sold by NEB), decreased
the steps and time involved, as a simple shift in
temperature inactivates the enzyme prior to the
ligation step (25).
DNA sequencing arrives. DNA sequencing was
developed in the late 1970s when two competing
methods were devised. Maxam and Gilbert
developed the “chemical sequencing method,”
which relied on chemical modification of DNA
and subsequent cleavage at specific bases (26). At
the same time, Sanger and colleagues published
on the “chain-termination method”, which became
2007
Introduction of ligationindependent
cloning (35)
1990
00
1985
2010
1995
1987
First automated
sequencers
become available
1983
Introduction
of PCR (28)
20
20
05
1993
Mullis shares the
Nobel Prize for his
work in PCR
1985
Generation of
pUC plasmids
(21)
2000
in vitro synthesis
of a whole genome
(41,42)
2008
Synthesis of the
mycoplasma genitalium
genome using Gibson
Assembly® (39)
&
Introduction of
High-Fidelity (HF®)
restriction enzymes
2010
First synthetic life –
Mycoplasma mycoides
(45)
5
feature article continued…
In 1970, Temin and Baltimore independently discovered reverse transcriptase in viruses, an enzyme
that converts RNA into DNA (29,30). Shortly after PCR was developed, reverse transcription was
coupled with PCR (RT-PCR) to allow cloning of
messenger RNA (mRNA). Reverse transcription
was used to create a DNA copy (cDNA) of mRNA
that was subsequently amplified by PCR to create an insert for ligation. For their discovery of
the enzyme, Howard Temin and David Baltimore
were awarded the 1975 Nobel Prize in Medicine
and Physiology, which they shared with Renato
Dulbecco.
the method used by most researchers (27). The
Sanger method quickly became automated, and
the first automatic sequencers were sold in 1987.
The ability to determine the sequence of a stretch
of DNA enhanced the reliability and versatility of
molecular cloning. Once cloned, scientists could
sequence clones to definitively identify the correct recombinant molecule, identify new genes or
mutations in genes, and easily design oligonucleotides based on the known sequence for additional experiments.
The impact of the polymerase chain reaction. One of
the problems in molecular cloning in the early
years was obtaining enough insert DNA to clone
into the vector. In 1983, Mullis devised a technique that solved this problem and revolutionized
molecular cloning (28). He amplified a stretch of
target DNA by using opposing primers to amplify
both complementary strands of DNA, simultaneously. Through cycles of denaturation, annealing and polymerization, he showed he could
exponentially amplify a single copy of DNA. The
polymerase chain reaction, or PCR, made it possible to amplify and clone genes from previously
inadequate quantities of DNA. For this discovery,
Kary Mullis shared the 1993 Nobel Prize in
Chemistry “for contributions to the developments
of methods within DNA-based chemistry”.
Cloning of PCR products. The advent of PCR meant
that researchers could now clone genes and DNA
segments with limited knowledge of amplicon
sequence. However, there was little consensus as to
the optimal method of PCR product preparation
for efficient ligation into cloning vectors.
Several different methods were initially used for
cloning PCR products. The simplest, and still the
most common, method for cloning PCR products
is through the introduction of restriction sites
onto the ends of the PCR product (31). This
allows for direct, directional cloning of the insert
into the vector after restriction digestion. Bluntended cloning was developed to directly ligate
PCR products generated by polymerases that
produced blunt ends, or inserts engineered to have
Figure 2. Overview of PCR
Reaction Assembly
3´ DNA region
5´ of interest
5´
3´
Amplification reaction
components are combined
and added to the thermocycler
98°C
5´
Denaturation
Temperature is increased
to separate DNA strands
3´
3´
5´
48 to 72°C
5´
Annealing
Temperature is decreased
to allow primers to base pair
to complementary DNA template
5´
3´
Primer
3´
3´
Primer
3´
5´
5´
Template
DNA strands
68 to 72°C
Extension
Polymerase extends primer
to form nascent DNA strand
Exponential
Amplification
Process is repeated,
and the region of interest
is amplified exponentially
1st cycle
5´
3´
3´
5´
5´
3´
3´
5´
2nd cycle
3rd cycle
4th cycle
22 = 4 copies
23 = 8 copies
24 = 16 copies
25 = 32 copies
6
Nascent
DNA strands
30th cycle
231 = 2 billion copies
restriction sites that left blunt ends once the insert
was digested. This was useful in cloning DNA
fragments that did not contain restriction sites
compatible with the vector (32).
Shortly after the introduction of PCR, overlap
extension PCR was introduced as a method to
assemble PCR products into one contiguous DNA
sequence (33). In this method, the DNA insert is
amplified by PCR using primers that generate a
PCR product containing overlapping regions with
the vector. The vector and insert are then mixed,
denatured and annealed, allowing hybridization of
the insert to the vector. A second round of PCR
generates recombinant DNA molecules of insertcontaining vector. Overlap extension PCR enabled
researchers to piece together large genes that
could not easily be amplified by traditional PCR
methods. Overlap extension PCR was also used to
introduce mutations into gene sequences (34).
Development of specialized cloning techniques.
In an effort to further improve the efficiency of
molecular cloning, several specialized tools and
techniques were developed that exploited the
properties of unique enzymes.
TA Cloning. One approach took advantage of a
property of Taq DNA Polymerase, the first heatstable polymerase used for PCR. During amplification, Taq adds a single 3´ dA nucleotide to the end
of each PCR product. The PCR product can be
easily ligated into a vector that has been cut and
engineered to contain single T residues on each
strand. Several companies have marketed the technique and sell kits containing cloning vectors that
are already linearized and “tailed”.
LIC. Ligation independent cloning (LIC), as its
name implies, allows for the joining of DNA
molecules in the absence of DNA ligase. LIC is
commonly performed with T4 DNA Polymerase,
which is used to generate single-stranded DNA
overhangs, >12 nucleotides long, onto both the
linearized vector DNA and the insert to be cloned
(35). When mixed together, the vector and insert
anneal through the long stretch of compatible
ends. The length of the compatible ends is sufficient to hold the molecule together in the absence
of ligase, even during transformation. Once transformed, the gaps are repaired in vivo. There are
several different commercially available products
for LIC.
USER cloning. USER cloning was first developed
in the early 1990s as a restriction enzyme- and
ligase-independent cloning method (36). When
first conceived, the method relied on using PCR
primers that contained a ~12 nucleotide 5´ tail, in
which at least four deoxythymidine bases had been
substituted with deoxyuridines. The PCR product
www.neb.com
was treated with uracil DNA glycosidase (UDG)
and Endonuclease VIII, which excises the uracil
bases and leaves a 3´ overlap that can be annealed
to a similarly treated vector. NEB sells the USER
enzyme for ligase and restriction enzyme independent cloning reactions.
In DNA assembly, blocks of DNA to be assembled
are PCR amplified. Then, the DNA fragments
to be assembled adjacent to one another are
engineered to contain blocks of complementary
sequences that will be ligated together. These
could be compatible cohesive ends, such as those
used for Gibson Assembly, or regions containing
Future Trends
recognition sites for site-specific recombinases
Molecular cloning has progressed from the clon(Gateway). The enzyme used for DNA ligation
ing of a single DNA fragment to the assembly of
will recognize and assemble each set of compatible
multiple DNA components into a single contiguous regions, creating a single, contiguous DNA
stretch of DNA. New and emerging technologies
molecule in one reaction.
seek to transform cloning into a process that is as
Synthetic biology. DNA synthesis is an area of
simple as arranging “blocks” of DNA next to each
synthetic biology that is currently revolutionizing
other.
recombinant DNA technology. Although a comDNA assembly methods. Many new, elegant tech- plete gene was first synthesized in vitro in 1972
nologies allow for the assembly of multiple DNA
(40), DNA synthesis of large DNA molecules did
fragments in a one-tube reaction. The advantages
not become a reality until the early 2000s, when
of these technologies are that they are standardresearchers began synthesizing whole genomes in
ized, seamless and mostly sequence independent.
vitro (41,42). These early experiments took years
In addition, the ability to assemble multiple DNA
to complete, but technology is accelerating the
fragments in one tube turns a series of previously
ability to synthesize large DNA molecules.
independent restriction/ligation reactions into a
Conclusion
streamlined, efficient procedure.
In the last 40 years, molecular cloning has proDifferent techniques and products for gene
gressed from arduously isolating and piecing toassembly include SLIC (Sequence and Ligase
gether two pieces of DNA, followed by intensive
Independent Cloning), Gibson Assembly (NEB),
screening of potential clones, to seamlessly assem®
GeneArt Seamless Cloning (Life Technologies)
bling up to 10 DNA fragments with remarkable
®
and Gateway Cloning (Invitrogen) (35,37,38).
efficiency in just a few hours, or designing DNA
Figure 3. Overview of the Gibson Assembly Cloning Method
DNA Preparation
+
Linear vector
B
A
DNA inserts with 15-20 bp
overlapping ends (PCR-amplified)
Gibson Assembly
A
Single-tube reaction
• Gibson Assembly Master Mix:
– Exonuclease chews back
5´ ends to create singlestranded 3´ overhangs
– DNA polymerase fills
in gaps within each
annealed fragment
– DNA ligase seals nicks
in the assembled DNA
B
Assembled
DNA
Incubate at 50°C
for 15-60 minutes
Transformation
DNA Analysis
OR
RE Digest
OR
Colony PCR
molecules in silico and synthesizing them in vitro.
Together, all of these technologies give molecular
biologists an astonishingly powerful toolbox for
exploring, manipulating and harnessing DNA,
that will further broaden the horizons of science.
Among the possibilities are the development of
safer recombinant proteins for the treatment of
diseases, enhancement of gene therapy (43), and
quicker production, validation and release of new
vaccines (44). But ultimately, the potential is constrained only by our imaginations.
Rebecca Tirabassi is an Assistant Editor at Bitesizebio.com.
References
1. Bertani, G. and Weigle, J.J. (1953) J. Bacteriol. 65, 113–121.
2. Luria, S.E. and Human, M.L. (1952) J. Bacteriol. 64, 557–569.
3. Linn, S. and Arber, W. (1968) Proc. Natl. Acad. Sci. USA 59, 1300–1306.
4. Smith, H.O. and Wilcox, K.W. (1970) J. Mol. Biol. 51, 379–391.
5. Danna, K. and Nathans, D. (1971) Proc. Natl. Acad. Sci. USA 68, 2913–
2917.
6. Kellenberger, G., Zichichi, M.L. and Weigle, J.J. (1961) Proc. Natl. Acad. Sci.
USA 47, 869–878.
7. Meselson, M. and Weigle, J.J. (1961) Proc. Natl. Acad. Sci. USA 47, 857–868.
8. Bode, V.C. and Kaiser, A.D. (1965) J. Mol. Biol. 14, 399–417.
9. Cozzarelli, N.R., Melechen, N.E., Jovin, T.M. and Kornberg, A. (1967)
Biochem. Biophys. Res. Commun. 28, 578–586.
10.Gefter, M.L., Becker, A. and Hurwitz, J. (1967) Proc. Natl. Acad. Sci. USA 58,
240–247.
11.Gellert, M. (1967) Proc. Natl. Acad. Sci. USA 57, 148–155.
12.Olivera, B.M. and Lehman, I.R. (1967) Proc. Natl. Acad. Sci. USA 57, 1426–
1433.
13.Weiss, B. and Richardson, C.C. (1967) Proc. Natl. Acad. Sci. USA 57, 1021–
1028.
14.Jackson, D.A., Symons, R.H. and Berg, P. (1972) Proc. Natl. Acad. Sci. USA
1972, 69, 2904–2909.
15.Griffith, F. (1928) J. Hyg. 27, 113–159.
16.Avery, O.T., Macleod, C.M. and McCarty, M. (1944) J. Exp. Med. 1944, 79,
137–158.
17.Mandel, M. and Higa, A. (1970) J. Mol. Biol. 1970, 53:159–162.
18.Cohen, S.N., Chang, A.C. and Hsu, L. (1972) Proc. Natl. Acad. Sci. USA 69,
2110–2114.
19.Cohen, S.N., Chang, A.C., Boyer, H.W. and Helling, R.B. (1973) Proc. Natl.
Acad. Sci. USA 70, 3240–3244.
20.Bolivar, F. et al. (1977) Gene 2, 95–113.
21.Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33, 103–119.
22.Roberts, R.J., Vincze, T., Posfai, J. and Macelis, D. (2010) Nucleic Acids Res.
38, D234–D236.
23.Mossner, E., Boll, M. and Pfleiderer, G. (1980) Hoppe Seylers Z. Physiol. Chem.
361, 543–549.
24.Green, M. and Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual,
(4th ed.), (pp. 189–191). Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
25.Rina, M. et al. (2000) Eur. J. Biochem. 267, 1230–1238.
26.Maxam, A.M. and Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA 74, 560–
564.
27.Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA
74, 5463–5467.
28.Mullis, K.B. and Faloona, F.A. (1987) Methods Enzymol. 155, 335–350.
29.Baltimore, D. (1970) Nature 226, 1209–1211.
30.Temin, H.M. and Mizutani, S. (1970) Nature 226:1211–1213.
31.Kaufman, D.L., and Evans, G.A. (1990) Biotechniques 9, 304, 306
32.Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Science 233, 1076–1078.
33.Horton, R.M. et al. (1989) Gene 77, 61–68.
34.Ho, S.N. et al. (1989) Gene 77, 51–59.
35.Li, M.Z. and Elledge, S.J. (2007) Nat. Methods 4, 251–256.
36.Nisson, P.E., Rashtchian, A. and Watkins, P.C. (1991) PCR Methods Appl. 1,
120–123.
37.Gibson, D.G. et al. (2009) Nat. Methods 6, 343–345.
38.Hartley, J.L., Temple, G.F. and Brasch, M.A. (2000) Genome Res. 10, 1788–
1795.
39. Gibson, D.G. et al. (2008) Science 319, 1215–1220.
40.Agarwal, K.L. et al. (1970) Nature 227, 27–34.
41.Blight, K.J., Kolykhalov, A.A. and Rice, C.M. (2000) Science 290 1972–
1974.
42.Couzin, J. (2002) Science 297, 174–175.
43.Hackett, P.B., Largaespada, D.A. and Cooper, L.J. (2010) Mol. Ther. 18,
674–683.
44.Dormitzer, P.R. et al. (2013) Sci. Transl. Med. 5, 185ra68.
45.Gibson, D.G. et al. (2010) Science 329, 52–56.
Sequencing
7
Cloning Reagents / Technical Guide
From traditional to advanced molecular cloning techniques, NEB has the right solution for you. Our high-quality reagents are available for every step in
the workflow, whether it be specialized enzymes, competent cells or novel solutions – such as Gibson Assembly. Educational tools and technical support
are available to you each step of the way, ensuring that you can clone with confidence. When you think of cloning, think NEB!
The cloning workflow figure below is an excerpt from the new NEB Cloning Brochure and Cloning Technical Guide, respectively, and compares the various
cloning methodologies. To learn more about each of these workflows, as well as the portfolio of products available from NEB, request for a free personal copy
at your local distributor or visit CloneWithNEB.com.
INSERT PREPARATION
VECTOR PREPARATION
Starting
Material
OR
OR
Plasmid
gDNA
Traditional Cloning
(RE Digestion & Ligation)
OR
Annealed oligos
PCR Cloning
(TA & Blunt-End)
cDNA
Seamless Cloning
(Gene Assembly)
OR
PCR
90 min.
RE Digestion
60 min. (Standard)
5–15 min. (Time-Saver)
DNA
Preparation
cDNA Synthesis
OR
PCR products
OR
Multiple cloning site
(MCS)
Starting
Material
RNA
Plasmid
OR
LIC (Ligation
Independent Cloning)
PCR
90 min.
OR
Recombinational
(Gateway/Creator/Univector)
Restriction Enzyme
(RE) Digestion
PCR
90 min.
PCR
90 min.
P
dsDNA
intermediate
P
OR
Dephosphorylation
Blunting
(Optional)
DNA End
Modifications
OR
A
A
P
30 min.
Clean Up
15 min.
Clean Up
15 min.
Phosphorylation
(Optional)
30 min.
Cohesive-End
Formation by
5´ → 3´ exo
30 min.
Cohesive-End
Formation by
3´ → 5´ exo
30 min.
OR
P
P
OR
P
Vector & Insert
Joining
P
P
Gel and Column
Purification
75 min.
Ligation
Instant – 15 min.
OR
T
A
OR
Recombination
sites
1 hr., 20 min. – 3 hr.
T
T
OR
Linear vector,
ready for joining
Proteinase K Treatment
10 min.
Estimated total time
20 min. – 2 hr., 25 min.
3 hr., 45 min.
70 min.**
A
T
OR
Holding vector
Assembled vector
Estimated total time*
T-addition
1.5 hr.
Clean Up 15 min OR
Gel & Column Purification 75 min.
Site-Specific
Recombination
60 min.
Annealing
30 min.
Dephosphorylation (Optional)
30 min.
DNA End
Modifications
+
Clean Up
15 min.
P
Ligation
Occurs simultaneously
with previous step
P
Linear vector
P
Ligation
15 min.
PCR
2 hr.
Clean Up
15 min.
OR
RE Digest
60 min. (Standard)
5–15 min. (Time-Saver)
A
A
P
Clean Up
15 min.
A
A
Clean Up
15 min.
P
dsDNA
intermediate 2
OR
A
A
PCR
P
P
OR
P
OR
RE Digestion
60 min. (Standard)
5–15 min. (Time-Saver)
DNA
Processing
2 hr. – 2 hr., 30 min.
2 hr., 15 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
2 hr., 45 min.
Endpoint vector
3 hr., 15 min. – 5 hr., 20 min.
DNA Isolation
(Plasmid Purification)
DNA
Analysis
** 70 minutes for recombination occurs on second day.
Protein
Expression
OR
RE Digest
Functional
Analysis
OR
Colony PCR
Sequencing
Site-Directed
Mutagenesis
Request
your
personal copy
from your local
distributor!
TOOLS & RESOURCES
Reagents and Tools for
Molecular Cloning
• Learn about recommended products for
cloning in our “Reagents and Tools for
Molecular Cloning” Brochure.
• Order your copy now at your local
distributor!
8
Molecular Cloning
TECHNICAL GUIDE
• Our Cloning Technical Guide contains
product selection charts, protocols, tips
for optimization and troubleshooting.
• Order your copy now at your local
distributor!
www.neb.com
Easy & effective ligation for all end types
– now at special discount
Once your insert and vector are end-treated, as needed, and have compatible ends, you’ll be ready to
ligate. NEB offers an extensive selection of high-quality and performance-optimized DNA ligases and
ligase master mixes; you’ll be certain to find an enzyme specific for your end type, enabling maximum
efficiency. Since time is always at a premium in the lab, you can speed up your reaction setup and incubation by choosing one of our ligase master mixes. With so many ligases to choose from, make NEB
your source for DNA ligases.
Top DNA
Ligases at
Special Prices!
offer closes
31.05.2014
ll
ll
ll
lll
Ligation of blunt ends
l
lll
ll
ll
lll
T/A cloning
l
lll
ll
ll
ll
lll
ll
Quick Ligation Kit
(#M2200)
lll
T4 DNA Ligase
(#M0202)
Blunt/TA Ligase
Master Mix (#M0367)
Ligation of sticky ends
ElectroLigase®
(#M0369)
Instant Sticky-end Ligase
Master Mix (#M0370)
DNA Ligase Selection Chart for Cloning
DNA APPLICATIONS
Electroporation
Repair of nicks
in dsDNA
High complexity
library cloning
lll
lll
lll
lll
ll
ll
ll
lll
Contact your local distributor for details!
recommended products
• For the ligation of sticky ends, we
recommend our Instant Sticky-end
Ligase Master Mix (#M0370)
or the Quick Ligation™ Kit
(#M2200)
or the ligation of blunt ends, we
• F
recommend our Blunt/TA Ligase
Master Mix (#M0367) or the
Quick Ligation Kit (#M2200)
he Blunt/TA Ligase Master Mix
• T
(#M0367) is recommended for T/A
cloning
lll
Blunt/TA Ligase Master Mix outperforms
the competition
Ligation in 15 min. or less


Master Mix Formulation


Recombinant







Ordering Information
PRODUCT
NEB #
SIZE
Instant Sticky-end Ligase Master Mix
M0370S/L
50/250 rxns
Blunt/TA Ligase Master Mix
M0367S/L
50/250 rxns
ElectroLigase
M0369S
50 rxns
T4 DNA Ligase
M0202S/L*
20,000/100,000 u
Quick Ligation Kit
M2200S/L
30/150 rxns
*also available as 5x concentrated versions
Transformation efficiency (%)
(as compared to Blunt/TA Ligase Master Mix)
FEATURES
100
T/A ligation
Blunt ligation
75
50
25
0
Company
A
Company
B
Company
NEB
C
Blunt/TA Ligase
Master Mix
Duplicate ligation reactions of blunt or T/A vector/insert pairs
were set up according to the master mix vendors’ suggestions.
Equal amounts of ligated DNA were used to transform NEB 10beta Competent E. coli (NEB #C3019) and triplicate plating was
performed. Transformation results were averaged and graphed
as a percentage of the highest performing reaction, T/A ligation,
using the Blunt/TA Ligase Master Mix.
9
New Cloning Tools to
Enhance Your Research
NEW!
Gel Loading Dye, Purple (6X)
ADVANTAGES
The new Gel Loading Dye, Purple (6x) is a unique and novel buffer/dye formulation for loading any
DNA sample onto agarose electrophoresis gels: it results in brighter and sharper bands compared to
“standard” bromophenol blue/glycerol-based loading buffers!
This new purple gel loading dye does not cast any “UV shadow” and therefore does
not quench or interfere with the UV-signal deriving from your DNA bands.
Our recommendation:
• Unique dye/buffer formulation
for tighter, brighter bands in DNA
Agarose Gels
• Unique purple dye migrates at same
speed as bromophenol blue
• Dye does not interfere with UV-light
(casts no UV-shadow)
For best electrophoresis results, use the new Gel Loading Dye, Purple (6x) for all
your agarose DNA gels!
Added Value!
Also available:
2-Log Quick-Load® Purple DNA Ladder (see back cover).
Now, free tube of
Gel Loading Dye, Purple
with all HF restriction
enzymes!
Ordering Information
PRODUCT
NEB #
SIZE
Gel Loading Dye, Purple (6X)
B7024S
4 ml
www.neb.com/HF
NEW! Cloning of “unstable” DNA inserts
– NEB Stable™ Competent E. coli
NEB Stable Competent E. coli is the ideal strain for cloning “difficult” unstable inserts, such as direct
or inverted repeats. It is the recommended host strain for cloning genes into retroviral/lentiviral
vectors. NEB Stable Competent E. coli offers high efficiencies and is competitively priced.
NEB Stable enables the isolation of plasmid clones containing repetitive DNA elements
A.
LU
Stable 5xREP insert (S)
623 bp
B.
L
S
UU
S
UU SSU–U
S
U
S
U UU
L = TriDye 100 bp
DNA Ladder
(NEB #N3271)
U = Unstable insert
with deletions
S = Stable 5xREP insert
623 bp – = No insert detected
Ordering Information
PRODUCT
NEB #
SIZE
NEB Stable Competent E. coli
C3040H
20 rxns
™
10
ADVANTAGES
• Improved cloning of direct repeats and
inverted repeats
• Recommended host strain for cloning
genes into retroviral/lentiviral vectors
• Free of animal products
• Value pricing
Plasmid pUC-5xREP contains five 32 bp repeats, making it unstable
in a recombination-proficient strain.
(A) NEB Stable competent cells or (B) Stbl3 competent cells
(Invitrogen) were transformed with 2 µl of a pUC-5xREP Gibson
Assembly reaction containing 2.2 ng (0.00125 pmol) pUC19 vector
and approximately 1 ng (0.0028 pmol) 5xREP insert. Transformed
cultures were plated on LB plates containing 100 µg/ml ampicillin
and incubated overnight at 30°C. The next day, colony PCR was
performed using M13/pUC polylinker primers to analyze 5xREP
insert stability. This figure shows the results of analyzing 33
independent colonies.
NEW!
Your PCR Cloning Kit from NEB
The NEB PCR Cloning Kit allows quick and simple cloning of all your PCR amplicons, regardless
of the polymerase used as it works equally well with blunt-end and TA end PCR products!
The kit utilizes a unique 2x Cloning Master Mix with a proprietary ligation enhancer that
efficiently combines blunting and ligation with no purification step! The ready-to-use novel
“suicide” cloning vector contains a toxic minigene for extremely efficient background colony
suppression.
For excellent results and convenience, optimized and ready-to-use competent E. coli cells
are already included in the kit. Enjoy faster cloning with more flexible conditions: clone with
any amplicon made with any DNA polymerase, with or without 5´ phosphates, purified or not!
PCR cloning with no background
ADVANTAGES
• Easy cloning of all PCR products,
including blunt and TA ends
• Fast cloning experiments with
5-minute ligation step
• Simplified screening with
low/no colony background and
no blue/white selection required
• Save time by eliminating
purification steps
• Flanking restriction sites available
for easy subcloning, including choice
of two single digest options
• Provided analysis primers allow for
downstream colony PCR screening
or sequencing
A 500 bp PCR product incubated with the linearized vector in a
3:1 ratio according to recommended protocol. 2 µl of reaction
was transformed into provided NEB 10-beta Competent E. coli
and 1/20th of the outgrowth was plated. The left plate serves
as the control, with vector backbone only. The right plate
contains PCR insert.
• Ready-to-use kit components include
1 kb control amplicon, linearized
cloning vector and single-use
competent E. coli cells
Top
New
Product!
Ordering Information
PRODUCT
NEB #
SIZE
NEB PCR Cloning Kit*
E1202S
20 rnxs
*including competent E.coli cells.
NEW!
recombinant Shrimp Alkaline Phosphatase
DNA phosphorylation is a key step in the cloning workflow. For your next cloning experiment,
consider Shrimp Alkaline Phosphatase (rSAP). rSAP is a heat labile alkaline phosphatase purified
from a recombinant source. rSAP contains no affinity tags or other modifications.
rSAP heat inactivation at 65°C
90
• Active in all NEB restriction enzyme
buffers: just add rSAP to restriction
digest
80
Remaining Activity (%)
• Recombinant quality with significantly
improved consistency compared to
native enzyme
• Completely and irreversibly heat
inactivated in just 5 min at 65°C
100
70
60
50
• Does not require supplemental
additives such as zinc, as is the case
with Antarctic Phosphatase
40
30
20
10
0
ADVANTAGES
0
10
5
15
Incubation at 65°C, minutes
20
1 unit of rSAP was incubated under recommended
reaction conditions, including DNA, for 30 minutes, and
then heated at 65°C. Remaining phosphatase activity
was measured by PNPP assay.
Ordering Information
PRODUCT
NEB #
recombinant Shrimp Alkaline Phosphatase (rSAP) M0371S/L
SIZE
500/2500 units
11
featured products
Competent Cells from NEB for Cloning
ADVANTAGES
• High transformation efficiencies
Selecting a competent cell line for cloning requires that you strike the right balance; you want a strain
that offers high transformation efficiencies, but also has the ability to generate sufficient plasmid
yields. NEB cloning strains offer high transformation efficiencies, and are T1 phage resistant and endA
deficient, ensuring high-quality plasmid preparation. Additionally, RecA– strains minimize potential
plasmid recombination. For convenience, NEB cloning strains are available in a variety of formats,
including single-use tubes and 96-well plates. Electrocompetent and subcloning efficiencies are also
available. Your choice of competent cell line for cloning can influence the success of your cloning
experiment; NEB offers a cell line for every need.
• Convenient formats
• Includes SOC media
• T1 phage resistance
• Free of animal products
Competent Cell Selection Chart
FEATURES
Versatile
l
l
Fast growth (< 8 hours)
l*
Toxic gene cloning
l
l
l
Large plasmid/
BAC cloning
l
Dam/Dcm free
plasmid growth
l
Retroviral/lentiviral
vector cloning
l
FORMATS
Chemically Competent
l
l
Electrocompetent
l
l
Subcloning
l
l
l
l
l
l
* RecA+
Ordering Information
12
PRODUCT
NEB #
SIZE
NEB 5-alpha Competent E. coli
C2987H/I/P
20/24 rxns/1x96 wells
NEB Turbo Competent E. coli
C2984H/I
20/24 rxns
NEB 5-alpha F´ Iq Competent E. coli
C2992H/I
20/24 rxns
NEB 10-beta Competent E. coli
C3019H/I
20/24 rxns
dam–/dcm– Competent E. coli
C2925H/I
20/24 rxns
NEB Stable Competent E. coli
C3040H
20 rxns
Benefit from the high transformation
efficiencies of NEB 5-alpha.
Transformation Efficiency (x109)
(cfu/µg pUC19)
NEB
NEB 5-alpha NEB Turbo 5-alpha F´ I q NEB 10-beta dam –/dcm –
NEB Stable
Competent
Competent
Competent
Competent
Competent
Competent
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
(NEB #C2987) (NEB #C2984) (NEB #C2992) (NEB #C3019) (NEB #C2925) (NEB #C3040)
2.5
2.0
1.5
1.0
0.5
0
NEB 5-alpha
MAX Efficiency®
DH5α™-T1R
The transformation efficiencies of NEB 5-alpha and DH5α were compared
using each manufacturers’ recommended protocols. Values shown are the
average of triplicate experiments.
NEB
Quick Cloning Box-HF:
Essential Cloning
Reagents for only
599,- CHF!
only 599,- CHF
This offer is valid until 31.05.2014.
No other discounts apply.
All prices in CHF excl. VAT.
Please mention promotion code
NES0412 when ordering.
featured product
Gibson Assembly Cloning Kit
New England Biolabs has revolutionized your laboratory’s standard cloning methodology. The Gibson
Assembly Cloning Kit combines the power of the Gibson Assembly Master Mix with NEB 5-alpha
Competent E. coli, enabling fragment assembly and cloning in just under two hours. Save time, without
sacrificing efficiency.
Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute,
and licensed to NEB by Synthetic Genomics, Inc., and allows for successful assembly of multiple DNA
fragments, regardless of fragment length or end compatibility.
Gibson Assembly efficiently joins multiple overlapping DNA fragments in a single-tube isothermal reaction.
The Gibson Assembly Master Mix includes three different enzymatic activities that perform in a single
buffer:
• The exonuclease creates a single-stranded 3´ overhang that facilitates the annealing of fragments that
share complementarity at one end
• The polymerase fills in gaps within each annealed fragment
• The DNA ligase seals nicks in the assembled DNA
Resulting DNA is ready to be transformed.
ADVANTAGES
• Rapid cloning into any vector with no
additional sequence added
• Easy-to-use protocols enable
cloning and transformation in just
under two hours
• High efficiencies, even with assembled
fragments up to 20 kb
• Includes competent cells
• Use NEBuilder®, our online primer
design tool, to design primers for your
Gibson Assembly reaction
Benefit from our free video tutorials at
www.neb.com
Making ends meet is now quicker and easier than ever before, with the Gibson Assembly Cloning Kit
from NEB.
Fragment Assembly
Ordering Information
PRODUCTS
NEB #
SIZE
Gibson Assembly Cloning Kit
E5510S
10 reactions
Gibson Assembly Master Mix
E2611S/L
10/50 reactions
NEB 5-alpha Competent E. coli
C2987H
C2987I
C2987P
20 x 0.05 ml
6 x 0.2 ml
1 x 96 well plate (20 ml/well)
NEB 10-beta Competent E. coli
C3019H
C3019I
20 x 0.05 ml
6 x 0.2 ml
For optimal PCR-Primer design
for Gibson Assembly, please use
the NEBuilder online tool at
http://nebuilder.neb.com
RELATED PRODUCTS
14
Some components of this product are manufactured by New England
Biolabs, Inc. under license from Synthetic Genomics, Inc.
www.neb.com
application note
Robust Colony PCR from Multiple
E. coli Strains using OneTaq
Quick-Load Master Mixes
Yan Xu, Ph.D., New England Biolabs, Inc.
GENERAL PROTOCOL
1. Transform ligation mix or other
plasmid-containing reaction mixture
into the desired bacterial strain, and
incubate agar plates overnight at the
appropriate temperature.
2. Set up 50 µl reactions as follows:
Introduction
OneTaq Master Mix
Colony PCR is a commonly used method to quickly screen for plasmids containing a desired insert
directly from bacterial colonies. This method eliminates the need to culture individual colonies and
prepare plasmid DNA before analysis. However, the presence of bacterial cell contents and culture
media in colony PCR reactions often results in polymerase inhibition. A robust polymerase is required
to perform colony PCR with high efficiency in many different bacterial strains.
OneTaq DNA Polymerase has been formulated for robust yields with minimal optimization. This
robustness makes OneTaq ideal for use in demanding applications, such as colony PCR. (For more
information on OneTaq DNA Polymerase, visit www.neb.com/OneTaq)
Results
Transformation of a plasmid containing a 4.5 kb insert into eighteen different competent cell lines was
performed, followed by colony PCR using either the OneTaq or OneTaq Hot Start Quick-Load 2X
Master Mix with Standard Buffer. Similar results were obtained using OneTaq and OneTaq Hot Start
(OneTaq data not shown), and the 4.5 kb insert was successfully amplified in each case.
Colony PCR of a 4.5 kb insert using OneTaq Hot Start
Quick-Load 2X Master Mix and 18 different E. coli strains
M12345 67891011
121314151617
18
4.5 kb
25 µl
PCR primer
200 nM
Nuclease-free water
to 50 µl
ote: If OneTaq Hot Start Quick-Load 2X Master Mix is
N
used, reactions can be set up at room temperature. If OneTaq
Quick-Load 2X Master Mix is used, reactions should be set
up on ice.
3. Use a sterile toothpick to pick up
individual colonies and dip into each
reaction tube.
4. A
s soon as the solution looks cloudy,
remove the toothpick. To create a
stock of each individual colony either:
Dip the toothpick into 3 ml growth
media with appropriate antibiotics
and culture overnight.
or
Streak the toothpick onto another
agar plate containing the appropriate
antibiotics and grow overnight.
5. T
ransfer reactions to a thermocycler,
and perform PCR following the
guidelines below for cycling
conditions:
Reactions were set up according to the protocol and analyzed by agarose electrophoresis. Marker M is the 1 kb DNA Ladder (#N3232S/L)
Summary
94°C
OneTaq and OneTaq Hot Start Quick-Load Master Mixes provide reliable performance in colony
PCR, and are compatible with multiple E. coli strains. Reliable performance has been seen with amplicons up to 10 kb (data not shown). The Quick-Load format offers additional convenience by enabling
direct loading of the PCR reaction onto an agarose gel for analysis. Lastly, the Hot Start formulation
provides additional functionality by reducing interference from primer-dimers and secondary amplification products.
Ordering Information
PRODUCT
NEB #
SIZE
OneTaq® DNA Polymerase
M0480S/L/X
200/1,000/5,000 u
OneTaq® 2X Master Mix with Standard Buffer
M0482S/L
100/500 rxns
OneTaq 2X Master Mix with GC Buffer
M0483S/L
100/500 rxns
OneTaq® Quick-Load® 2X Master Mix with Standard Buffer
M0486S/L
100/500 rxns
®
OneTaq Quick-Load 2X Master Mix with GC Buffer
M0487S/L
100/500 rxns
OneTaq® Hot Start DNA Polymerase
M0481S/L/X
200/1,000/5,000 u
OneTaq® Hot Start 2X Master Mix with Standard Buffer
M0484S/L
100/500 rxns
OneTaq® Hot Start 2X Master Mix with GC Buffer
M0485S/L
100/500 rxns
®
OneTaq Hot Start Quick-Load 2X Master Mix with Standard Buffer
M0488S/L
100/500 rxns
OneTaq® Hot Start Quick-Load® 2X Master Mix with GC Buffer
M0489S/L
100/500 rxns
®
®
INITIAL DENATURATION
®
2 minutes
30 CYCLES
94°C
15–30 seconds
45–68°C
15–60 seconds
68°C
1 minute per kb
FINAL HOLD
68°C
5–10 minutes
10°C
hold
6. Load 4 – 6 µl of each PCR reaction
directly onto an agarose gel, alongside
an appropriate DNA ladder.
The full Application Note can be downloaded using the QR code above or at
www.neb.com/OneTaq
15
DNA CLONING
DNA AMPLIFICATION & PCR
EPIGENETICS
RNA ANALYSIS
LIBRARY PREP FOR NEXT GEN SEQUENCING
PROTEIN EXPRESSION & ANALYSIS
CELLULAR ANALYSIS
www.neb.com
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938-2723
Top
DNA Ladders
at
Built
for
Perfect
Migration!
Special Prices!
offer closes
31.05.2014
New!
kb ng
10.0 42
8.0 42
6.0 50
5.0 42
New!
kb ng
4.0 33
3.0 125
2.0 48
1.5 36
1.0 42
3
100
2
37
ng
45
1.200
35
70
58
54
50
46
42
76
34
31
27
95
27
24
700
21
250 57
600
18
200 107
1
400
33
916
766
700
650
600
550
500
450
400
350
1,000
900
800
500/517 97
0.5 42
0,8% TAE agarose gel.
Mass Values are for
0,5 µg/lane
bp
1.517
1.5 27
0.5 33
1 kb DNA Ladder
*
bp
ng
1.350 103
48.5 30
20 30
15 45
10 33
8 33
6 40
5 33
4 26
300 46
150 46
38
0,6% TBE agarose gel.
Mass Values are for
0,5 µg/lane
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
0.7
0.6
122
34
31
27
23
0.5
124
0.4
49
0.3
37
300
29
200
25
0.2
32
100
48
0.1
61
100 69
50
Quick-Load® 1 kb
Extend DNA Ladder
Your local NEB distributor:
100 bp DNA Ladder
1,3% TAE agarose gel.
Mass Values are for
0,5 µg/lane
2-Log DNA Ladder
1,0% TBE agarose gel.
Mass Values are for
1 µg/Lane
1,8% TBE agarose gel.
Mass Values are for
1 µg/Lane
DNA CLONING
DNA AMPLIFICATION & PCR
EPIGENETICS
RNA ANALYSIS
PRODUCT
NEB#
SIZE
1 kb DNA Ladder
N3232S/L
200/1000 gels
1 kb TriDye DNA Ladder
N3272S
SAMPLE PREP FOR NEXT GEN SEQUENCING
PROTEIN EXPRESSION & ANALYSIS
CELLULAR ANALYSIS
Built for
Perfect Migration.
Magellanic Penguin (Spheniscus magellanicus) migrate
northwards during winter to find adequate food resources.
125/375 gels
DNA Ladders
kb
ng
10,0
8,0
6,0
5,0
4,0
42
42
50
42
33
3,0
125
2,0
48
1,5
36
1,0
0,5
kb
45
1,200
35
1,000
900
800
700
600
95
27
24
21
18
100
37
500/517
97
1,5
27
400
38
1
33
300
29
200
25
0,5
33
100
48
1 kb Extend
Quick-Load® DNA Ladder
0,6% TBE agarose gel.
Mass values are for
0,5 µg/lane.
NEB #N3239
ng
2,0
40
1,5
57
1,2
45
1,0
0,9
0,8
0,7
0,6
122
34
31
27
23
0,5
124
0,4
49
0,3
37
0,2
32
0,1
61
N3236S/L
50 bp Quick-Load® DNA Ladder
N0473S
2-Log DNA Ladder
N3200S/L
2-Log TriDye DNA Ladder
N3270S
N0469S/L
2-Log Quick-Load® DNA Ladder
2-Log Quick-Load® Purple
New! N0550S
DNA Ladder*
70
58
54
50
46
42
76
34
31
27
300
46
250
57
200
107
150
46
100
69
50
ng
42
500
27
350
300
250
20
33
27
200
110
150
33
100
43
75
58
50
63
25
43
50 bp DNA Ladder
1,8 % TBE agarose gel.
Mass values are for 1 µg/lane.
NEB #N3236
1
bp
ng
766
62
500
40
300
48
150
61
2
kb
ng
kb
ng
10,0
8,0
45
45
10,0
6,0
240
180
6,0
5,0
45
136
4,0
120
3,0
75
4,0
3,5
3,0
2,5
45
45
45
45
2,0
60
2,0
45
1,0
45
kb
kb
ng
10,0
6,0
4,0
3,0
15
30
45
60
2,0
120
1,0
180
0,5
300
10.0
5.0
3.0
2.0
1.5
1.0
0,766
0,500
0,300
0,150
50
2-Log DNA Ladder
1,0 % TBE agarose gel.
Mass values are for 1 µg/lane.
NEB #N3200
DNA Ladders in “ready-to-use” formats
bp
766
84
100 bp DNA Ladder
– for your convenience
Low Molecular Weight
DNA Ladder
1,8% TBE agarose gel. Mass
values are for 0.5 µg/lane.
NEB #N3233
New!
89
PCR Marker
0,5
Supercoiled DNA Ladder
1,8% TBE agarose gel.
Mass values are for
0,3 µg/lane.
NEB #N3234
0,8 % TAE agarose gel.
Mass values are for 0,5 µg/lane.
NEB #N0472
30
Mass DNA Ladder
1 % TBE agarose gel.
Mass values are for
0,75 µg/lane.
NEB #N3237
0,050
Reverse Mass DNA Ladder
0,8 % TAE agarose gel.
Mass values are for
0,75 µg/lane.
NEB #N3240
Quick-Load® DNA Ladders “ready-to-use” prediluted in gel loading buffer
containing blue or purple tracking dye.
125 gels
8.454
7.242
6.369
5.686
4.822
4.324
bp
bp
TriDye™ 1 kb DNA Ladder NEB #N3272
TriDye™ 100 bp DNA Ladder NEB #N3271
TriDye™ 2-Log DNA Ladder NEB #N3270
1.353
2.323
4.361
pBR322 DNA –
BstNI Digest
1,4 % TBE agarose gel.
NEB #N3031
310
281
271
234
194
118
1.371
2.027
Lambda DNA –
HindIII Digest
Lambda DNA –
BstEII Digest
1,0 % TBE agarose gel.
NEB #N3014
125/375 gels
67
250 gels
200/1000 gels
250 gels
250/750 gels
250 gels
*NEW: Also available as Quick-Load Purple format. See page 10 for details.
1,5
63,5
48,5
33,5
2,32
1,7 % TBE agarose gel.
NEB #N3026
1,8 % TBE agarose gel.
NEB #N3032
bp
1.000
500
300
500
bases
Low Range
PFG Marker
25
21
17
500
microRNA Marker
60 ng (5 µl) on 12 %
denaturing polyacrylamide-urea gel.
SYBR Gold staining
NEB #N2102
1.900 + 1.640
945
915
815
785
745
680
242,5
610
555
194
145,5
450
375
295
97
225
Midrange I PFG Markers
1 % agarose gel. 6 V/cm,
15 °C for 20 h. Switch times
ramped from 0,5-1,5 sec.
NEB #N3019
1 µg/lane. 1 % agarose gel.
6 V/cm, 15 °C for 15 h. Switch
times ramped from 1–12 sec.
NEB #N0350
1% agarose gel, 6 V/cm, 15°C for
24 hours. Switch times ramped
from 1-25 seconds.
NEB #N3551
48,5
Yeast Chromosome
PFG Marker
Lambda Ladder
PFG Marker
1 µg/lane. 1 % agarose gel.
6 V/cm, 15 °C for 26 h. Switch
times of 70 sec for 15 h and
120 sec for 11 h.
NEB #N0345
1 µg/lane. 1 % agarose gel.
4,5 V/cm, 15 °C for 48 h. Switch
times ramped from 5-120 sec.
NEB #N0340
150
80
80
50
50
30
21
Low Range
ssRNA Ladder
kDa
175
175
66,4
80
80
58
58
46
46
2,0 % TBE agarose gel.
1 µg heated at 60 °C for
5 min prior to loading
NEB #N0364
bp ng
42,7
25
21
17
34,6
15
15
15
dsRNA Ladder
siRNA Marker
1,5 µg on 2.0 % TBE
agarose gel.
NEB #N0363
45 ng (5 µl) on 20 % TBE
polyacrylamide gel.
NEB #N2101
27,0
30
kDa
30
25
20,0
kDa
kDa
230
150
230
150
100
100
100
80
80
80
60
60
60
50
50
40
40
30
30
50
40
30
25
150
25
FINLAND:
BIONORDIKA OY
Kutomotie 18
00380 Helsinki
Tel: +358/207/410 270
Fax +358/207/410 277
[email protected]
www.bionordika.fi
HUNGARY:
KVALITEX KFT
Pannónia u. 5.
1136 Budapest
Phone: (1) 340-4700
Fax: (1) 339-8274
[email protected]
www.kvalitex.hu
25
23
20
14,3
17
6,5
7
Protein Marker, Broad
Range (2 - 212 kDa)*
10-20 % SDS-PAGE
NEB #P7702
*Note that 2,3 and 3,4 kDa
bands run at the dye front
www.neb.com
kDa
250
212
158
116
97,2
55,6
300
150
2.000
ssRNA Ladder
727,5
679
630,5
582
533,5
485
436,5
388
339,5
291
kb
1.120 + 1.100
15
Lambda DNA –
Mono Cut Mix
kDa
1,0 % TBE agarose gel.
1 µg heated at 60 °C for
5 min prior to loading
NEB #N0362
200/1000 gels
97
82
Protein Markers
bases
5.000
1.000
112
4,36
23,1
2,03
pBR322 DNA –
MspI Digest
RNA Markers
3.000
242,5
227,5
209
194
179
160,5
145,5
130,5
6,55
48,5
10,1
76
φX174 DNA –
HaeIII Digest
kb
kb
9,42
97
17,1
15
90
72
1.264
1,0 % TBE agarose gel.
NEB #N3012
194
145,5
242 + 238
217
201
190
180
160 + 160
147 + 147
123
110
603
1.929
2.322
48,5
38,4
33,5
29,9
24,5
24
307
1.078
872
6.557
1.058
929
kb
404
bp
9.416
1.857
kb
622
527
3.675
23.130
bp
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
ISRAEL:
ORNAT BIOCHEMICALS AND
LABORATORY EQUIPMENT LTD.
POB 2071
Rehovot 76120
Tel: +972-8-9477077
Fax: +972-8-9363934
[email protected]
www.ornat.co.il
TriDye™ DNA Ladders “ready-to-use” prediluted
in gel loading buffer containing xylene cyanol FF,
bromophenol blue and orange G (runs at
approximately 50 bp in regular 1 % TBE gels).
Quick-Load® 1 kb DNA Ladder NEB #N0468
Quick-Load® 1 kb Extend DNA Ladder NEB #N3239
Quick-Load® 100 bp DNA Ladder NEB #N0467
Quick-Load® 2-Log DNA Ladder NEB #N0469
Quick-Load® Purple 2-Log DNA Ladder NEB #N0550
Quick-Load DNA Ladders
during electrophoresis.
lane 1:
Quick-Load 2-log DNA Ladder
lane 2:
Quick-Load Purple 2-log DNA Ladder
Fast DNA Ladder*
1,2 % TBE agarose
gel. Mass values are
for 0,5 µg/lane.
NEB #N3238
bp
100/500 gels
7.000
50 bp DNA Ladder
103
916
766
700
650
600
550
500
450
400
350
New: For even brighter and sharper bands, NEB offers a new purple gel-loading buffer (#B7024S)
– optimal for use in combination with HF® Restriction Enzymes.
The Quick-Load® Purple 2-log DNA Ladder (#N0550) already contains the new loading buffer!
9.000
N0467S/L
ng
1.350
1,3 % TAE agarose gel.
Mass values are for
0,5 µg/lane.
NEB #N3231
bases
100 bp Quick-Load® DNA Ladder
bp
Conventional DNA & PFG Markers
121
N3271S
ng
1,517
3
42
1 kb DNA Ladder
kb
2
383
100 bp TriDye DNA Ladder
ng
48,5 30
20
30
15
45
10
33
8
33
6
40
5
33
4
26
42
0,8 % TAE agarose gel.
Mass values are for
0,5 µg/lane.
NEB #N3232
125 gels
kb
10,0 40
8,0 40
6,0 48
5,0 40
4,0 32
3,0 120
New!
* Fast DNA Ladder can be used for fast electrophoresis systems as well as standard electrophoresis.
N0468S/L
1 kb Quick-Load DNA Ladder
1 kb Extend Quick-Load®
New! N3239S
DNA Ladder
100 bp DNA Ladder
N3231S/L
®
125 gels
CZECH REPUBLIC:
BIOTECH A.S.
Sluzeb 4
108 52 Praha 10
Tel: (Toll Free) 0800 124683
Fax: 02 72701742
[email protected]
www.biotech.cz
GREECE:
BIOLINE SCIENTIFIC
1 Meg. Alexandrou Str.
10437 Athens
Tel: 210 5226547
Fax: 210 5244744
[email protected]
84
50 bp DNA Ladder
BeNeLux:
BIOKÉ
Schuttersveld 2
2316 ZA Leiden
The Netherlands
Tel: +31(0)71 568 1000
Tel: Belgium: 0800 71640
Fax: +31(0)71 568 1010
[email protected]
www.bioke.com
17
7
Prestained Protein
Marker, Broad Range
(7 - 175 kDa)
ColorPlus Prestained
Protein Marker, Broad
Range (7 - 175 kDa)
10-20 % SDS-PAGE
NEB #P7708
10-20 % SDS-PAGE
NEB #P7709
15
10
Protein Ladder
(10 - 250 kDa)
10-20 % SDS-PAGE
NEB #P7703
20
20
15
15
10
10
Prestained Protein
Ladder, Broad Range
(10–230 kDa)
ColorPlus Prestained
Protein Ladder, Broad
Range (10–230 kDa)
10–20 % SDS-PAGE
NEB #P7710
10–20 % SDS-PAGE
NEB #P7711
[email protected]
RZ_pinguine_NEB_Marker-Poster_D_F_EU.indd 1
New England Biolabs GmbH, August 2013,
Für Druckfehler keine Haftung; Printed in Germany
©
07.01.14 10:36
Order your free copy of
the new Electrophoresis
Poster.
Contact your local distributor
for details.
Printed in Germany
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/
NORWAY:
BioNordika Norway AS
P.O. Box 298
1326 Lysaker
Tel +47 67 11 14 60
Fax +47 67 11 14 61
[email protected]
www.bionordika.no
POLAND:
Lab-JOT Ltd. Sp.z o.o.
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
SWEDEN:
BIONORDIKA SWEDEN AB
Box 21160
S-100 31 Stockholm
Tel: (08) 30 60 10
Fax: (08) 30 60 15
[email protected]
www.bionordika.se
SPAIN/PORTUGAL:
IZASA S.A. Plaza de Europa, nº 21-23
08908 L’Hospitalet de Llobregat
(Barcelona)
Tel: 902.20.30.70
Fax: 902.20.30.71
[email protected]
www.izasa.com
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
2.CAD.ATB IS MERKEZI C
BLOK NO:52
Macunkoy/ANKARA 06370
Tel:+903123977746-47
Fax:+903123977748
[email protected]
www.sacem.com.tr