Cloning Tools
Cloning tools
product selection guide
A researcher’s guide to cloning DNA
Cloning Tools
You’ll find it all here
→ Innovative kits and reagents that help reduce the time for cloning and simplify your procedures
→ Achieve reliable, reproducible performance at every stage of your cloning experiment
→ Comprehensive brochure to take the guesswork out of product selection
One resource for all your cloning needs
Whether you’re looking for your gene of interest, already possess your starting nucleic acid, or are a few steps into your cloning experiment,
this guide will help you successfully complete your cloning experiment. It contains a wealth of information about the products we offer for
your cloning protocols. To make it easier to navigate, this brochure is divided into five sections, each focusing on a different aspect of the
overall cloning procedure. We begin with a brief introduction, move into different ways of obtaining starting materials, present various cloning
methods and technologies, give an overview of downstream procedures, and finish with custom cloning services.
Contents
Introduction
A cloning overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Getting started
What cloning method is best for me? . . . . . . . . . . . . . . . . . 12
Choosing a cloning method: TOPO® Cloning . . . . . . . . . . 13
Where do I start?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Choosing a cloning method: Gateway® technology . . . 15
Working with your starting material:
purchase a clone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Choosing a cloning method: restriction enzyme
digestion and ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Working with your starting material:
obtain clones from cDNA libraries . . . . . . . . . . . . . . . . . . . . . . 4
Working with your starting material:
purify RNA or DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Working with your starting material:
reverse-transcribe RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Working with your starting material:
amplify DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Working with your starting material:
purify PCR products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2
Cloning technologies
Downstream procedures
What happens after the cloning reaction? . . . . . . . . . . . . . 19
After the cloning reaction: transformation . . . . . . . . . . . . . 19
After the cloning reaction: plasmid purification . . . . . . . 22
After the cloning reaction: electrophoresis . . . . . . . . . . . . 24
Custom services
Custom DNA oligos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Let the experts do the cloning for you . . . . . . . . . . . . . . . . 27
Introduction
A cloning overview
We offer a number of proven technologies, kits, and reagents to help you achieve your cloning
goals. The sections in this guide provide additional information about each choice to help determine which path is the best for your experiments. Keep in mind that there are often several solutions that will work equally well with every challenge.
Where do I start?
No matter what starting source is available, you will need genomic
Once you have your DNA, you’ll probably need to amplify it to
material to begin the cloning process (Figure 1). This section dis-
generate a sufficient amount for the actual cloning reaction.
cusses the various ways to prepare or obtain ready-to-clone DNA,
We offer a number of amplification enzymes, each optimized
including:
for use with different DNA challenges such as fidelity, sensitiv-
→
Purchasing a premade clone
ity, yield, length, and GC-rich content. The most popular DNA
→
Reverse-transcribing from RNA
polymerases are discussed on page 8. For complete informa-
→
Purifying from tissues or cells
tion on these enzymes and our entire PCR enzyme offering, visit
→
Obtaining from a library
www.invitrogen.com/pcr.
Starting
marterial
Cloning
Downstream
procedures
Library
Functional
analysis
Tissues/cells
DNA isolation
PCR and
clean-up
RNA isolation
Cloning
Transformation
Plasmid
purification
DNA analysis
RT-PCR
Protein
expression
Clone
Figure 1. Cloning workflow.
3
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Cloning Tools
Working with your starting material:
purchase a clone
blast, accession numbers, or clone IDs. You can also use the Gene
Ontology classification to browse the entire collection. Each clone
Ultimate™ human and mouse open reading frames
(ORFs)
has an Ultimate™ ORF Card that brings together all the informa-
→
Time-saving tools—full-length, sequence-verified ORFs
symbol, Gene Ontology, SNPs, and detailed protein annotation.
eliminate the tedious procedures of RNA isolation, cDNA
The Ultimate™ ORF Clone Collection is updated quarterly with
synthesis, and sequence validation
the most current information from the GenBank®, Ensembl, and
Reliable quality—guaranteed to match the corresponding
Swiss-Prot databases. We frequently monitor the collection so
reference sequence in the GenBank® database at the amino
that if any Ultimate™ ORF Clone becomes invalid, it is deactivated
acid level*
and moved to the ORFanage Collection.
→
→
tion you need on each ORF clone, including splice variants, gene
Comprehensive selection—over 16,000 clones available
for important gene families like kinases, nuclear hormone
receptors, cytokines, and transcription factors (just to name
a few)
Working with your starting material: obtain
clones from cDNA libraries
Construct highly representative cDNA libraries with
the CloneMiner™ II cDNA Library Construction Kit
The easiest way to obtain your gene of interest is to purchase
→
High primary titers
it directly from our Ultimate™ ORF Clone Collection. The Ulti-
→
Large average insert sizes, typically >1.5 kb
mate™ Human and Mouse ORF Clone Collection offers full-insert
→
Highest percentage of full-length genes
sequenced clones in a Gateway® vector for direct access to virtually unlimited downstream applications. See pages 15–17 to learn
The innovative CloneMiner™ II library construction technology
more about Gateway® technology.
combines SuperScript® III Reverse Transcriptase with Gateway®
technology to produce highly representative cDNA libraries with-
Find your ORF today
out restriction enzyme cloning. This enables the discovery of pre-
Visit www.invitrogen.com/cloning and select Clone Collections to
viously unobtainable, fully intact clones. The kit also avoids the
see what Ultimate™ ORF Clones are available. Our bioinformatics tool
use of lengthy and inefficient ligation reactions, making library
will allow you to search our collections using keywords, sequence
construction faster and offering more complete representation.
Ordering information
Product
Quantity
Ultimate™ ORF Clones
Order online at www.invitrogen.com/cloning.
CloneMiner™ II cDNA Library Construction Kit
5 cDNA rxns
*Nucleotide sequence may vary.
4
Cat. No.
A11180
Working with your starting material:
purify RNA or DNA
tamination. The PureLink™ RNA Mini Kit provides rapid columnbased purification of total RNA from a wide range of cell and tissue
RNA and DNA isolation kits designed to produce high yields
of total RNA (Table 2) or DNA from cells or tissues
types.
→
Obtain high RNA yields with the most trusted and widely cited
of only a few steps of pipetting and magnetic separation, with
TRIzol® RNA reagent (Molecular Research Center)
all operations performed in a single tube. Contaminating PCR
Achieve midiprep capacity in a convenient miniprep format with
inhibitors are eliminated without any centrifugation steps or use
the PureLink™ RNA Mini Kit
of phenol/chloroform. The kit is particularly useful for isolation of
Rapid 10-minute protocol helps provide high-quality PCR-ready
DNA from bacteria and cultured cells, small samples, as well as
gDNA, even from small samples with the Dynabeads® DNA
from clinical specimens and tissues from various species. Proto-
DIRECT™ Kit
cols have also been developed for fully automated use on liquid-
→
→
The Dynabeads® DNA DIRECT™ Universal protocol consists
Starting material
handling robots.
PureLink™ technology combines nontoxic lysis with the speed, purity,
The PureLink™ Genomic DNA Mini Kit and PureLink™ Pro 96
and ease of use of silica-membrane column RNA purification. The safe
Genomic DNA Kit enable high-yield, high-purity DNA extractions
and easy RNA purification procedure can be completed in less than
from a wide variety of sample types, including blood, tissues,
20 minutes, and high-quality total RNA can be obtained from mini- to
cells, bacteria, swabs, and blood spots, in a familiar silica spin col-
midiprep amounts of starting material with no genomic DNA con-
umn or semi-skirted 96-well plate format. A large range of sample
sizes can be used with the PureLink™ Genomic DNA Kits.
Table 2. Advantages of selected RNA purification technologies.
Product
Chemistry
Advantages
TRIzol® Plus RNA Purification System
Phenol/chloroform,
silica spin column
•
•
•
•
PureLink™ RNA Mini Kit and Pro 96
Total RNA Purification Kit
Silica spin column
• High binding capacity (1,000 μg)
• Easy to use, familiar, 20-minute protocol
• Scalable—only 1 kit needed for all sample sizes
Integrates sample lysis with TRIzol® Reagent and spin-column RNA purification
Offers an unmatched 1,000 μg binding capacity in a miniprep format
Eliminates time-consuming isopropanol precipitation
Industry-recommended protocol for gene expression analysis
Ordering information
Product
Quantity
Cat. No.
TRIzol® Plus RNA Purification System
50 preps
12183-555
PureLink™ RNA Mini Kit
50 preps
12183-018A
PureLink™ Pro 96 Total RNA Purification Kit
4 x 96 preps
12173-011A
Dynabeads® DNA DIRECT™ Kit
300 preps
630-06
PureLink™ Genomic DNA Mini Kit
50 preps
K1820-01
PureLink™ Pro 96 Genomic DNA Purification Kit
4 x 96 preps
K1821-04A
To learn more about RNA or DNA purification products, visit www.invitrogen.com/naprep.
5
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Cloning Tools
Working with your starting material:
reverse-transcribe RNA
Enabling accurate gene representation with highperforming SuperScript® III Reverse Transcriptase (RT)
High-yield one-step RT-PCR for cloning
→
High cDNA yields—greater thermostability provides a long
System with Platinum® Taq High Fidelity
half-life of typically 220 min at 50°C
→
Superior product yields from targets up to 9 kb (Figure 2)
More full-length cDNA—50°C reaction temperature helps
→
Sensitive detection from as little as 1 pg HeLa total RNA
overcome RNA secondary structure
→
Simple one-tube, one-step reaction format
Increased specificity with gene-specific primers—retains
→
Direct compatibility with multiple applications, including
→
→
full activity at 50°C
Complete RT-PCR system—SuperScript® III One-Step RT-PCR
multiplex RT-PCR and cloning
Choose SuperScript® III RT for robust first-strand synthesis and great
The SuperScript® III One-Step RT-PCR System with Platinum® Taq
cDNA yields. Engineered to be RNase H minus and thermostable up
High Fidelity provides high product yields and sequence fidelity
to 55°C, SuperScript® III RT delivers best-in-class cDNA synthesis in
for applications such as cloning, gene detection, and quantifica-
every single reaction, combining maximum yield with maximum
tion. The enhanced thermostability of SuperScript® III RT enables
sensitivity. You get full-length cDNA without degrading rare RNA
superior cDNA yields, while the addition of Platinum® Taq DNA
transcripts. SuperScript®III RT always delivers the results you need to
Polymerase High Fidelity helps deliver full-length, high-fidelity PCR.
move forward—the first time, every time. Go for complete reliability at
www.invitrogen.com/superscript.
Figure 2. High fidelity and performance of the SuperScript® III One-Step RT-PCR System with Platinum® Taq High Fidelity. One-step RT-PCR reactions were performed according
to each manufacturer’s recommendations using the primers and starting HeLa total RNA quantities indicated. The thermostability and reduced RNase H activity of
SuperScript® III RT is important for tackling RNA with secondary structure. SuperScript® III RT provides higher yields of full-length cDNA compared to other reverse
transcriptase enzymes.
6
Starting material
Time-saving supermix
The SuperScript® III First-Strand Synthesis SuperMix is designed
to provide high yields of first-strand cDNA in a convenient highthroughput supermix format. The simple, time-saving reaction
setup uses just two tubes—a 2X reaction mix and a SuperScript® III
enzyme mix (Figure 3). An optional annealing buffer is provided for
improved yields and sensitivity. The Enzyme Mix includes SuperScript® III Reverse Transcriptase to enable higher cDNA yields,
superior sensitivity—down to 1 pg—and specificity, and compatibility with a wide variety of RT-PCR applications.
Figure 3. SuperScript® III First-Strand Synthesis SuperMix provides a convenient
time-saving reaction setup.
Ordering information
Product
Target size
Sensitivity
Specificity
Convenience
Quantity
Cat. No.
SuperScript® III Reverse
Transcriptase
≤12 kb
•••
•••
••
200 units
10,000 units
18080-093
18080-044
SuperScript® III One-Step
RT-PCR System with Platinum® Taq High Fidelity
≤9 kb
•••••
•••
•••••
25 rxns
100 rxns
12574-030
12574-035
SuperScript® III First-Strand
Synthesis SuperMix
≤12 kb
•••
•••
••••
50 rxns
18080-400
7
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Cloning Tools
Working with your starting material:
amplify DNA
How to choose between chemically modified and
antibody-mediated hot-start reagents
Proven DNA polymerases
Chemically modified hot-start reagents are best when perform-
→
High yield
ing high-throughput applications. These enzymes withstand lon-
→
High fidelity
ger periods at room temperature. However, the activation time is
→
High sensitivity
longer and the read lengths are shorter.
Antibody-mediated hot-start products deliver fast activation
No matter what the source of DNA, you will most likely need to
times and minimal template degradation, which promote the
use PCR to generate sufficient material for cloning. We offer best-
amplification of longer fragments. However, antibody-mediated
in-class PCR enzymes with brands you trust including our most
chemistries tend to exhibit reduced specificity and involve longer
popular enzymes for cloning (Table 3). For a complete list of our
setup times.
PCR enzymes and the benefits of using each, visit www.invitrogen.com/pcr.
With both types of hot-start technologies available from
Invitrogen, you can choose the chemistry that works best for your
research.
Hot-start amplification of a broad range of targets
DNA Polymerase
Robust PCR amplification with high yields and
increased fidelity
→
Increased specificity with choice of trusted hot-start
Platinum® Taq DNA Polymerase High Fidelity
technologies
→
AmpliTaq Gold® 360 DNA Polymerase (Roche) and Platinum® Taq
8
Greater than six times higher fidelity than Taq DNA
→
Amplify the broadest range of targets (<3 kb)
→
Available in Master Mix or SuperMix formats for increased
→
Amplification of fragments up to 20 kb
convenience
→
High yields with no need to optimize reaction conditions
polymerase
Chemically modified hot-start AmpliTaq Gold® 360 DNA Poly-
The accuracy of Platinum® Taq DNA Polymerase High Fidelity is
merase has been optimized to amplify a wide range of targets
provided by a mixture of Platinum® Taq DNA Polymerase and the
while delivering the high specificity, yield, and reliability you have
proofreading (3´ ➝ 5´ exonuclease activity) enzyme from Pyrococ-
come to expect.
cus species, GB-D polymerase. PCR specificity is improved with
Platinum® Taq DNA Polymerase uses antibody-mediated
the incorporation of Platinum® automatic “hot-start” technology.
hot-start to enable increased specificity, yield, and sensitivity with
Platinum® Taq DNA Polymerase High Fidelity is especially effec-
the fastest activation times.
tive for high-fidelity applications such as cloning or mutagenesis.
Although there are several types of hot-start enzymes, the
Platinum® Taq DNA Polymerase High Fidelity predominantly pro-
most reliable and widely used techniques involve chemically
duces PCR products with 3’-A overhangs for compatibility with
modified hot-start or antibody-mediated hot-start enzymes.
TOPO® TA Cloning® Kits.
Table 3. PCR enzyme selection guide.
Target size
Specificity
Fidelity
(vs. Taq)
Yield
Speed
Convenience
••••
<3 kb
••
1x
•••
••
••••
AmpliTaq Gold® 360 Master Mix
•••••
<3 kb
••
1x
••••
•••
•••••
Platinum® Taq DNA Polymerase
•••
<5 kb
••
1x
•••
••
••••
Platinum® Taq DNA Polymerase,
High Fidelity
•••
<20 kb
••
6x
••••
••
••••
AccuPrime™ Taq DNA Polymerase,
High Fidelity
•••
<20 kb
•••
9x
••••
••
••••
AmpliTaq Gold® 360 DNA Polymerase
Starting material
Amplify wide
target range
Product
Ordering information
Product
Quantity
Cat. No.
AmpliTaq Gold® 360 DNA Polymerase
100 units
250 units
4398813
4398823
AmpliTaq Gold® 360 Master Mix
1 mL
5 mL
4398876
4398881
Platinum® Taq DNA Polymerase
250 rxns
500 rxns
10966-026
10966-034
Platinum® PCR SuperMix
100 rxns
11306-016
Platinum® Taq DNA Polymerase, High Fidelity
100 rxns
500 rxns
11304-011
11304-029
Platinum® PCR SuperMix High Fidelity
100 rxns
12532-016
AccuPrime™ Taq DNA Polymerase, High Fidelity
200 rxns
1,000 rxns
12346-086
12346-094
10 mM dNTP Mix
100 μL
18427-013
100 mM dNTP Set
4 x 25 μL
10297-018
Since the introduction of our first thermal cycler in 1987, we have continued to develop and support
innovative Applied Biosystems® PCR instruments to advance your research. More information on our
latest thermal cycler—the Veriti® Thermal Cycler—can be found at www.appliedbiosystems.com.
9
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Cloning Tools
Working with your starting material:
purify PCR products
PureLink™ Quick Gel Extraction and PCR Purification Combo Kits
offer the ability to perform both a gel extraction or a PCR purifi-
Begin your cloning with only the PCR products you want
cation a single kit. The PureLink™ Quick Gel Extraction and PCR
→
High recovery
Purification Combo Kit is designed to purify DNA fragments from
→
Clean products
agarose gels. The simple procedure uses a silica-based spin cartridge to purify DNA fragments from 40 bp to 10 kb in <30 min-
Some cloning methods, such as TOPO® Cloning, allow you to go directly
utes from TAE or TBE agarose of various percentages and melting
from PCR to clone without any additional purification steps. For those
points. The PCR purification protocol achieves rapid and efficient
applications that require PCR clean-up or validation of PCR results, there
removal of short primers, dNTPs, enzymes, short-failed PCR prod-
are two methods generally followed: PCR product isolation using a col-
ucts, and salts from PCR fragments >100 bp, typically in less than
umn, and gel purification from an agarose gel.
10 minutes. DNA isolated using the PureLink™ kit is free of proteins, dye, and agarose, and is ready to use for a variety of applica-
For quick and easy gel extraction or PCR purification of
DNA samples
tions, including automated fluorescent DNA sequencing, manual
PureLink™ Quick Gel Extraction Kit and PCR Purification Combo Kit
cloning, and labeling.
DNA sequencing, PCR, in vitro transcription, restriction mapping,
→
Purify DNA fragments from 40 bp to 10 kb in <30 min
→
High recovery due to high binding column capacity of up to
Gel Extraction Kit (Figure 4) is designed for fast purification of
15 μg of DNA
DNA fragments from TAE or TBE agarose or polyacrylamide gels
Works well with both vacuum and centrifugation protocols
of various percentages without the use of organic solvents.
→
If you simply require a kit for gel extraction, the PureLink™
Figure 4. The PureLink™ Gel Extraction Kit procedure is quick and easy. Gel fragments are first solubilized to release the DNA. The sample is then loaded on a PureLink™
spin column and isolated using a simple bind/wash/elute procedure. DNA fragments are recovered in TE buffer or water, ready to use in downstream applications.
10
Purify PCR products from a complex mixture of DNA
using silica-based membrane technology
PureLink™ PCR Purification Kits are designed to provide rapid and
PureLink™ PCR Purification Kits
PCR products, and salts from PCR reactions. Specialized binding
→
Purify fragments between 100 bp and 15 kb or remove by-
buffers allow for purification of PCR products between 100 bp
products <300 bp
and 15 kb or removal of by-products <300 bp. PCR products
→
Simple bind/wash/elute procedure
purified using the PureLink™ PCR Purification Kits demonstrate
→
Elute ready-to-use, pure PCR product in typically <10 min
outstanding performance in downstream applications such as
efficient removal of short primers, dNTPs, enzymes, short-failed
Starting material
automated fluorescent DNA sequencing, PCR, restriction mapping, and labeling.
Ordering information
Product
Quantity
Cat. No.
PureLink™ Quick Gel Extraction and PCR Purification Combo Kit
50 preps
K2200-01
PureLink™ Gel Extraction Kit
50 preps
K2100-12
PureLink™ PCR Purification Kit
50 rxns
250 rxns
K3100-01
K3100-02
PureLink™ Pro 96 PCR Purification Kit
4 x 96-well plates
K3100-96A
Making your own agarose gels? For high-quality agarose
Looking for a fast, sensitive alternative to pour-your-own
gels that resolve DNA and RNA fragments from 100 bp to
agarose gels? Learn more about E-Gel® EX precast agarose
>30 kb, try our UltraPure™ Agarose (page 25).
gel electrophoresis (page 24).
11
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Cloning Tools
What cloning method is best for me?
A number of different methods, technologies, and protocols are
Table 4 below can help you navigate your way through these
available for performing the actual cloning reaction. To choose
technologies. For more detailed information, see pages 12–18.
the one that best meets your needs, you will need to analyze the
current parameters of your experiment and plan ahead for any
Table 4. When to choose a particular cloning technology.
downstream procedures. This section discusses three highly efficient cloning technologies:
→
→
→
TOPO®
Cloning
Gateway®
technology
•
Following traditional protocols
TOPO® Cloning provides simple, convenient reactions typi-
Cloning for storage
•
cally in less than 5 min
Cloning for sequencing
•
Gateway® technology offers the greatest flexibility in vector
Multiple downstream
expression options
choices and downstream applications
High-efficiency cloning
•
•
Restriction digestion, phosphatase treatment, and ligase
Time-savings
•
•
cloning is an industry standard for molecular biologists
DNA fragment modification
Starting
marterial
Cloning
•
•
Downstream
procedures
Library
Functional
analysis
Tissues/cells
DNA isolation
PCR and
clean-up
RNA isolation
Cloning
Transformation
Plasmid
purification
DNA analysis
RT-PCR
Protein
expression
Clone
12
Restriction
digestion
Choosing a cloning method: TOPO® Cloning
How it works
For faster, more effective cloning of any PCR product,
use TOPO® Cloning
The key to TOPO® Cloning technology is the Vaccinia virus enzyme
topoisomerase I, which cleaves and rejoins DNA during viral replica-
→
Fast—5 min room-temperature reaction
tion. The enzyme specifically cleaves one DNA strand, enabling
→
Easy—no gel purification or post-PCR modifications needed
the DNA to unwind, then religates the ends of the cleaved strands
→
Efficient—up to 99% recombinants
and releases itself from the DNA. Each TOPO® vector is supplied
linearized with topoisomerase I covalently attached to the 3´
Our unique TOPO® Cloning technology is the most convenient,
phosphate group at each end. These topoisomerase I-activated
effective, and reliable method available for cloning short or long
TOPO® vectors circularize readily with high efficiency in the pres-
PCR products amplified using Taq or a proofreading polymerase.
ence of linear DNA fragments with compatible ends. Typically in
All it takes is 5 minutes to achieve up to 99% recombination effi-
only 5 minutes at room temperature, the ligation is complete and
ciency. Vectors incorporating TOPO® technology are available
the circular vector is ready for transformation (Figure 5). There’s
for all major molecular biology applications, including cloning,
no need for specially modified primers or to spend the time,
sequencing, in vitro transcription, and gene expression.
effort, and reagents required in traditional restriction enzyme
digestion and ligation reactions. In fact, with TOPO® Cloning you
can use PCR products directly from the amplification reaction†—
Cloning technologies
no clean-up steps are needed.
Perform PCR with Taq or a
proofreading polymerase
Add 1 μl of PCR reaction to
1 μl of TOPO® Cloning vector
Incubate 5 minutes at
room temperature
Perform transformation with
provided competent E. coli strain
Figure 5. The TOPO® Cloning protocol.
†
The TOPO® XL PCR Cloning Kit requires a 15-minute gel purification step.
13
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Cloning Tools
Clone Taq-amplified or blunt-end PCR products
For the easiest blunt-end cloning (Figure 7):
Whether you have amplified your DNA with Taq or a Taq-like
→
The pCR-Blunt II-TOPO® vector in the Zero Blunt® TOPO®
DNA polymerase to generate PCR products with 3´ A overhangs,
PCR Cloning Kits contains the ccdB gene to reduce back-
or have a blunt-ended DNA insert to clone, there’s a TOPO® vec-
ground; M13, T7, and SP6 priming sites for convenient
tor for you. Here we discuss the features and benefits of three
sequencing; and the same 5-minute benchtop liga-
of our most popular TOPO® vectors. For a complete list, visit
tion as the TOPO® TA Cloning® reaction, yielding ≥95%
www.invitrogen.com/topo.
recombinants.
For fast cloning of Taq-amplified PCR products (Figure 6):
→
The pCR™8/GW/TOPO® vector is designed for multiple
To view the full range of TOPO® products and to select
purposes, including rapid recombination into a variety of
the kit that’s right for you, visit www.invitrogen.com/topo.
Gateway® destination vectors, convenient sequencing with
Gateway® primer sites, robust selection in E. coli with spectinomycin resistance, and EcoRI sites flanking the PCR product
insertion site for easy excision of inserts
→
The pCRII-TOPO® vector has dual promoters, T7 and SP6, for
easy sequencing and in vitro transcription of the cloned DNA
fragment
Figure 6. TOPO® TA Cloning® method for Taq-amplified DNA.
Figure 7. Zero Blunt® TOPO® Cloning of blunt-end DNA.
14
Choosing a cloning method: Gateway® technology
>95%), allowing you to effectively eliminate the need for secondary
Take advantage of powerful recombinational cloning
for gene shuttling and protein expression with
Gateway® technology
sequencing or subcloning after the initial entry clone is made. It also
→
Enables consistent results throughout your studies—use the
research, there will be a Gateway® technology available to meet your
same clone from target identification to validation
needs (Table 5).
→
allows you to clone hundreds to thousands of genes at the same
time for high-throughput purposes. Whatever your current stage of
Allows you to save time and effort—no need to subclone or
sequence each clone
→
Streamline workflow processes and perform multiple
applications
→
Collaborate efficiently with other scientists using vectors and
clones compatible with Gateway® technology
Gateway® technology is a universal platform that enables you
to shuttle your gene of interest (GOI) to as many expression and
functional analysis systems as you need (Figure 8). Orientation
Cloning technologies
and reading frame are maintained with high efficiencies (typically
Figure 8. Rapidly move from one application to the next with Gateway® technology.
Table 5. Gateway® technology meets your needs across a broad range of applications.
Stage of research
Application
Gateway® products
Gene acquisition
Drug target identification
Ultimate™ ORF Clone collection
Sequencing
Entry and donor vectors
Cloning
Cloning and subcloning
Building clone and library collections
CloneMiner™ II cDNA Library Construction Kit
Gene delivery into challenging mammalian cell lines
ViraPower™ Expression Systems
Delivery
In vivo studies in animal model systems
Protein arrays
Protein production
Expressway™ Plus Expression System
Champion™ pET Expression System
Antibody or antigen production
BaculoDirect™ Expression System
Protein analysis
Function
pcDNA™ mammalian destination vectors
Interactions
Two-hybrid systems
Reporter assays
GeneBLAzer® technology
Localization
GFP and Lumio™-tagged destination vectors
RNAi
BLOCK-iT™ technology
Purification
His-tagged destination vectors
15
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Cloning Tools
How it works
Gateway® technology uses lambda phage–based site-specific
→
From a Directional TOPO® Cloning vector: The Directional
recombination instead of restriction endonucleases and ligase to
Gateway® TOPO® entry vectors pENTR™/D-TOPO® and
insert your GOI into an expression vector. The DNA recombination
pENTR™/TEV/D-TOPO® allow you to take advantage of fast,
enzyme mixtures that mediate the recombination reactions are the
efficient Directional TOPO® Cloning and universal Gateway®
foundation of Gateway® technology. Transferring a gene into a desti-
technology (Figure 9). With Directional TOPO® Cloning, you
nation vector is accomplished in just two steps.
can clone PCR products containing your gene of interest in
the sense orientation in a 5-minute benchtop ligation. Greater
Step 1: Clone your gene of interest into a Gateway®
entry vector
than 90% of the recombinants will contain a PCR product in
There are a number of ways to enter the Gateway® platform, including
recombination with any Gateway® destination vector to create
use of TOPO® Cloning vectors containing Gateway® att sites, PCR or
an expression clone. In addition, the pENTR™/TEV/D-TOPO®
restriction-based cloning into Gateway® entry vectors, or purchasing
vector carries a TEV protease cleavage site for producing native
an Ultimate™ ORF Clone already in a Gateway® vector (page 4).
proteins after expression.
→
From a TOPO® TA Cloning® vector: The pCR™8/GW/TOPO®
→
From a PCR or restriction cloning vector without TOPO®
vector offers 5-minute cloning of Taq-amplified PCR products
Cloning: pDONR™ and pENTR™ vectors accept PCR products
into a vector ready for use in the Gateway® system. It is
amplified with primers containing attB sequences or specific
designed for multiple purposes, including rapid recombination
restriction sites, respectively. The resulting clones are ready for
into a variety of Gateway® destination vectors, convenient
recombination with any Gateway® destination vector.
sequencing with Gateway® primer sites, robust selection in
→
From a purchased clone: You can also enter Gateway®
E. coli with spectinomycin resistance, and EcoRI sites flanking
technology with a ready-to-use clone from an extensive
the PCR product insertion site for easy excision of inserts.
group of clone collections. The Ultimate™ ORF Clone
pcDNA™
expression
vector
pcDNA™
expression clone
Figure 9. How TOPO® Cloning and Gateway® recombination work together.
16
the desired orientation. These vectors also contain att sites for
Entry clone
Additional
expresion clones
Ever-increasing expression options
Collection consists of high-quality, full-insert sequenced
A wide variety of Gateway® destination vectors are avail-
human and mouse open reading frames already cloned into
able for protein expression and functional analysis experi-
the pENTR™221 Gateway® entry vector enabling limitless
ments. From in vitro to bacterial to insect to mammalian to viral
downstream analysis capabilities. Collections of Gateway®
systems, there is a system made for your application. Learn more at
destination vectors supplied by a wide variety of research
www.invitrogen.com/gateway.
groups are also available within the scientific community.
Step 2: Construct the expression clone
Mix the entry clone containing your gene of interest with the appro-
Destination
vector
Expression
clone
LR Clonase® II
priate Gateway® expression vector (destination vector) and Gateway®
Trasform E. coli and
select Ampr colonies
LR Clonase® II enzyme mix. Site-specific recombination between the
att sites in each vector generates an expression clone with the gene of
Entry clone
By-product
interest recombined into the destination vector backbone, and a byproduct (Figure 10). Following transformation and selection in E. coli,
Figure 10. The Gateway® LR recombination reaction.
the expression clone is ready for expression in the appropriate host.
Cloning technologies
Ordering information
Product
Quantity
Cat. No.
pCR™8/GW/TOPO® TA Cloning® Kit with One Shot® Mach1™ T1R E. coli
20 rxns
K2520-20
pENTR™/D-TOPO® Cloning Kit with One Shot® Mach1™ T1R E. coli
20 rxns
K2435-20
20 rxns
K2525-20
R
pENTR™/TEV/D-TOPO® Cloning Kit with One Shot® Mach1™ T1 E. coli
Gateway® destination vectors
www.invitrogen.com/gateway
Ultimate™ ORF Clones
www.invitrogen.com/cloning
To learn more about Gateway® technology and the complete selection of Gateway® entry options and destination vectors, visit
www.invitrogen.com/gateway.
17
www.invitrogen.com
Cloning Tools
Choosing a cloning method:
restriction enzyme digestion and ligation
Enzymes for traditional cloning
→
→
Consistent—extensive, rigorous performance and quality
of enzymes needed to clone and subclone your gene of interest
testing
(Table 6) so you can achieve your goals in both simple and complex
Convenient—supplied with optimized Universal buffers
cloning projects. Furthermore, having high-quality DNA throughout the cloning process is critical to success. We recommend using
The traditional method for cloning DNA can be divided into three
SYBR® Safe DNA Gel Stain and the Safe Imager™ Blue-Light Transil-
main steps: digest, modify, and ligate DNA. Each step requires high-
luminator for safe DNA visualization without harming your sample.
quality, reliable enzymes to ensure success. We offer a number
For more information on the SYBR® Safe stain and the Safe Imager™
transilluminator, see page 24.
Table 6. A sample listing of the high-quality enzymes available for restriction/ligation cloning methods.
Product
Quantity
Cat. No.
BamHI
10,000 units
15201-031
BglII
2,000 units
15213-028
EcoRI
20,000 units
15202-021
EcoRV
10,000 units
15425-036
HindIII
10,000 units
15207-020
KpnI
5,000 units
15232-036
PstI
5,000 units
15215-023
PvuII
1,500 units
15412-018
SalI
1,000 units
15217-011
XbaI
10,000 units
15226-038
XhoI
10,000 units
15231-020
Calf Intestinal Alkaline Phosphatase (CIAP)
1,000 units
18009-019
Bacterial Alkaline Phosphatase (BAP)
2,500 units
18011-015
DNA Polymerase I (Klenow)
100 units
18012-021
T4 DNA Ligase
500 units
15224-025
E. coli DNA Ligase
100 units
18052-019
Digest
Modify
Ligate
To view the full list of enzymes and to use our restriction enzyme selection tool visit www.invitrogen.com/restrictionenzymes.
18
What happens after the cloning reaction?
characteristics of your DNA and any desired applications. The key
Once the DNA fragment is cloned into a vector, you will need to
elements to consider when choosing the best competent cells for
characterize and validate each clone:
your cloning experiment include genetic markers (Table 7), trans-
1.
Transform E. coli with your clone. This allows you to gener-
formation efficiency, and packaging format (Tables 8 and 9).
ate sufficient material for any downstream procedures,
whether they are QC checks or higher-level experiments.
2.
3.
Table 7. Genetic markers to look for when cloning.
Purify your DNA. Now that you have your clone in bacteria,
Genetic marker
Description
you will need to recover the DNA from the cells.
endA1
Mutation indicates endonuclease deficiency;
ensures good quality of miniprep DNA with
no degradation
recA1
Mutation indicates general recombination
deficiency; ensures insert stability and helps
prevent unwanted recombination between
insert and host; inserts are more stable in
recA1 than in recA13 hosts
mcrA, mcrBC, and mrr
Mutations allow for the efficient cloning
of methylated genomic DNA or methylated cDNA; strains without these markers
recognize foreign DNA through methylation
patterns and restrict it, resulting in underrepresented libraries
tonA
Mutation in this gene (also called fhuA)
prevents T1 and T5 phage infection; also
involved in iron transport
Analyze your results. With gel electrophoresis and restriction enzyme techniques, you can now confirm that you have
generated the correct clone.
The following pages provide brief overviews of the products available for procedures downstream of the cloning reactions. For more
information, please visit www.invitrogen.com/cloning.
After the cloning reaction: transformation
E. coli strains that get the job done
→
Ready to use—available as chemically competent or
Table 8. Convenient packaging formats of competent cells.
electrocompetent
Product
Convenient—many strains supplied in a single-use format
→
Efficient—transformation efficiencies ranging from >1 x 106
Advantages
• Single-use, 50 μL aliquots of competent
cells
One Shot® Competent Cells
to >3 x 1010 cfu/μg
transform E. coli to propagate sufficient quantities of your DNA for
ready for transformation. The strain you choose depends upon the
Starting
marterial
Cloning
• Transform directly in the tube
• No extra pipetting steps, no extra
tubes, and no efficiency-zapping
freeze-thaw cycles
Once you have completed the cloning reaction, you will need to
downstream experiments. We offer a variety of competent cells
Cloning technologies
→
MultiShot™ Competent
Cells
• Designed for medium- to highthroughput cloning
• Competent cells predispensed into
either a 96-well plate or a 96-stripwell
plate
Downstream
procedures
Library
Functional
analysis
Tissues/cells
DNA isolation
PCR and
clean-up
RNA isolation
Cloning
Transformation
Plasmid
purification
DNA analysis
RT-PCR
Protein
expression
Clone
19
www.invitrogen.com
Cloning Tools
OmniMAX™ 2 T1 Phage-Resistant (T1R) cells offer the highest
Use our most versatile chemically competent cells for
your everyday cloning needs—TOP10 and DH5α™ cells
transformation efficiency of any chemically competent cells with
TOP10 chemically competent cells are provided at a transformation
>5 x 109 transformants/μg pUC19.
efficiency of 1 x 109 cfu/μg and are ideal for high-efficiency cloning
The OmniMAX™ 2 T1R E. coli strain is perfect for use in all clon-
and plasmid propagation.
ing applications, including Gateway® technology. In addition to high
TOP10 chemically competent cells allow stable replication of
transformation efficiencies, it provides efficient transformation of
high-copy number plasmids. One Shot® TOP10 Kits are available with
highly methylated DNA due to the lack of E. coli K-12 restriction sys-
either chemically competent or electrocompetent E. coli to fit your
tems (mcrAΔ(mrr hsdRMS-mcrBC)). OmniMAX™ 2 T1R cells also carry
specific transformation needs. TOP10 E. coli are also available in the
the tonA genotype, which confers resistance to T1 and T5 phage
high-throughput MultiShot™ format.
infection. This protects your samples and minimizes the possibility of
downtime in your lab due to phage contamination.
Genotype of TOP10 cells:
F– mcrA Δ(mrr-hsdRMS-mcrBC) ф80lacZΔM15 lacX74 recA1
Genotype of OmniMAX™ 2 T1R cells:
araΔ139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
F´ {proAB+ lacIq lacZΔM15 Tn10(TetR) Δ(ccdAB)} mcrA
Δ(mrrhsdRMS-mcrBC) ф80(lacZ)ΔM15 Δ(lacZYA-argF) U169 endA1
DH5α™ cells are similar to the reliable TOP10 cells and designed
recA1 supE44 thi-1 gyrA96 relA1 tonA panD
for general cloning procedures. They are provided in a variety of
Cloning Unstable DNA
transformation efficiencies and packaging formats.
In addition to cells for routine cloning, Invitrogen offers a number of
High-efficiency cloning—MegaX DH10B™ T1R
Electrocomp™ and OmniMAX™ 2 T1R Chemically
Competent Cells
cell lines that have been optimized to achieve the highest performance in its noted application.
MegaX DH10B™ T1R Electrocomp™ cells are the highest-efficiency
Optimize your results when cloning unstable DNA with MAX
electrocompetent cells available (Figure 11), with a guaranteed 3-fold
Efficiency® Stbl2™ and Stbl3™ cells. MAX Efficiency® Stbl2™ cells are
greater number of colonies per transformation (>3 x 1010 cfu/μg of
high-efficiency chemically competent cells, specifically designed for
pUC DNA). They are ideal for highly demanding cloning experiments.
cloning unstable inserts. In addition to recA1, a unique set of genetic
markers allows for stable cloning of direct-repeat and retroviral
Table 9. Competent cells are available in a variety of formats.
Chemically
competent
Electrocompetent
Single-use
One Shot®
•
•
•
MultiShot™ StripWell
•
MultiShot™
•
Standard
•
Format
20
•
High-throughput
Volume
Advantage
50 μL
Transformation and
recovery in the same
tube
•
50 μL per well
12 strips of 8 tubes;
do as many or as few
reactions as needed
•
15 μL per well
96-well plates fit
automated format
100 μL, 200 μL,
or 500 μL
Economical option
4
cfu/μg pUC DNA (× 1010)
3.5
sequences and tandem-array genes. These cells are also available in
bulk format.
The Stbl3™ E. coli strain is designed for cloning direct repeats
found in lentiviral expression vectors. These cells reduce the frequency of unwanted homologous recombination between long
3
2.5
2
1.5
1
0.5
0
L
terminal repeats (LTRs) found in ViraPower™ Lentiviral and other ret-
S
G
MegaX
R
Figure 11. MegaX DH10B™ T1 Electrocomp™ cells consistently outperform the
competition. L: E. cloni® 10G SUPREME cells (Lucigen); G: GC10™ Thunderbolt™
cells (GeneChoice); S: ElectroTen-Blue® electroporation-competent cells
(Stratagene). This graph shows the high-efficiency results you would expect
to see using MegaX DH10B™ T1R Electrocomp™ cells compared to electrocompetent cells available from other suppliers.
roviral vectors. They are available in the convenient One Shot® format.
Fast growing chemically competent cells—Mach1™ T1
Phage-Resistant (T1R) Cells
Following transformation of Mach1™ T1 Phage-Resistant (T1R)
1.8
1.6
Cells, colonies are typically visible within eight hours of plating the
O.D. at 600 nm
1.4
transformation mix, enabling you to plate and pick colonies on the
same day (Figure 12). Further time savings can be obtained since
minipreps can be performed after only 4 hours of growth from an
Mach1™ T1R
XL10-Gold®
TOP10
XL1-Blue®
DH5_»2
1.2
1.0
0.8
0.6
0.4
overnight colony.
0.2
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
Time (hours)
R
Genotype of Mach1™ T1 cells:
Figure 12. Mach1™ T1R cells grow faster compared to other cloning strains. Mach1™
T1R (Invitrogen), XL10-Gold® (Stratagene), TOP10 (Invitrogen), XL1-Blue®
(Stratagene), and DH5α™ E. coli were transformed with pUC19 and grown
overnight. Each culture was then inoculated 1:50 into 50 mL of LB ampicillin
in 500 mL flasks. A600 was measured every 30 min for 6 hr. Mach1™ T1R cells
reach a useful OD (e.g., for miniprep analysis) significantly faster (4 hr) than
other strains.
ΔrecA1398 endA1 tonA ф80 ΔlacM15 ΔlacX74 hsdR(rk–mk+)
In addition to competent cells, a number of products
needed to complete your transformation are available,
including media, antibiotics, and cuvettes. For complete
information on transformation support products, visit
To view the full range of chemically competent or elec-
www.invitrogen.com/compcells.
trocompetent cells, visit www.invitrogen.com/compcells.
Ordering information
Product
(C)hemically or (E)lectro
competent
Quantity
Cat. No.
>1 x 109
Subcloning Efficiency™ DH5α™ Cells
>1 x 10
6
C
20 x 50 μL
C4040-03
MAX Efficiency® DH5α™ Cells
>1 x 109
C
4 x 500 μL
18265-017
C
5 x 200 μL
18258-012
One Shot® OmniMAX™ 2 T1 Cells
>5 x 10
9
C
20
C8540-03
MegaX DH10B™ T1R Electrocomp™
>3 x 1010
E
5 x 100 μL
C6400-03
MAX Efficiency® Stbl2™ Cells
>1 x 109
C
5 x 200 μL
10268-019
Stbl3™ Cells
>1 x 10
8
C
20 x 50 μL
C7373-03
ElectroMAX DH10B™T1R Cells
>1 x 1010
E
5 x 100 μL
11635-018
>1 x 109
C
20 x 50 μL
C8620-03
>1 x 109
C
1 x 96-well plate
C8696-01
One Shot® TOP10 Cells
R
One Shot® Mach1™ T1R Cells
R
MultiShot™ Stripwell Mach1™ T1 Cells
Downstream procedures
Transformation
efficiency*
* Transformants/μg pUC19 DNA.
21
www.invitrogen.com
Cloning Tools
After the cloning reaction: plasmid purification
High-purity, transfection-grade plasmid DNA
Efficient kits for obtaining your clone from bacterial
culture
PureLink™ HiPure Plasmid Filter Maxiprep Kits
→
High purity levels for all your downstream applications
→
Multiple scales and formats to help meet your throughput
→
without centrifugation or syringe pressure
→
needs
→
Reliable, reproducible results
Simple protocol for efficient clearing of bacterial lysates
Faster results due to sample clarification and column loading combined in a single, time-saving step
→
Reliable, high yields (850 μg from maxipreps)
PureLink™ HiPure Plasmid Filter Maxiprep Kits are designed to
Now that you have a bacterial culture growing, you will need a
isolate high purity plasmid DNA. The kits combine a unique bac-
plasmid purification method to screen for and obtain your clone.
terial lysate filtration system with a gravity-flow anion-exchange
We offer several kits that can help you accomplish this goal. A select
resin column to improve both the speed and performance of the
few are discussed below. For complete information on all of our
purification process. A patented resin combines increased surface
nucleic acid purification kits, visit www.invitrogen.com/naprep.
area and unique attachment chemistry to allow for greater yield
and reproducible performance. DNA purified using the PureLink™
Quick and easy purification of molecular biology–
grade plasmid DNA
HiPure Plasmid Maxiprep Kits is transfection-grade and can also
PureLink™ Quick Plasmid Miniprep Kit
modifications.
be used for in vitro transcription and translation, and enzymatic
→
Reproducible high yields up to 40 μg per spin column
→
Compatible design works with both vacuum and centrifu-
Endotoxin-free plasmid DNA in 15 minutes
gation formats
ChargeSwitch® Pro Filter Plasmid Kits
Rapid protocol provides ready-to-use DNA in minutes
→
→
PureLink™ Quick Plasmid Miniprep Kits are designed for rapid isolation of molecular biology–grade plasmid DNA from 1–5 mL of
scales (mini, midi, and maxi)
→
culture. PureLink™ spin columns make use of a unique silica membrane to help achieve the highest yields possible (up to 40 μg) fol-
Endotoxin-free plasmid DNA (0.01–0.1 EU/μg), equivalent to
competitor’s endo-free plasmid kits
→
Easy, hassle-free setup—100% ethanol-free, guanidinium-
lowing a simple bind/wash/elute procedure. Isolated DNA is free
free, organic solvent–free, aqueous protocol; complete
of RNA, proteins, and salt, and is ready to use in standard molecu-
ready-to-go kit with elution tubes provided
lar biology applications.
22
Fastest protocol—plasmid DNA in as little as 15 min for all
A
B
The new ChargeSwitch® Pro Filter kits, with the novel dual nested
column design, provide a fast and simple protocol for isolation
of endotoxin-free plasmid DNA (Figures 13 and 14). The nested
column design allows for simultaneous clarification of lysate and
binding of plasmid DNA in a single short centrifugation or vacuum step. The kits come as a complete ready-to-go package with
no additional steps of adding ethanol to wash buffers or drying
Figure 13. ChargeSwitch® Pro Filter Plasmid Kits. (A) ChargeSwitch® Pro Filter
columns (left to right): mini, midi, and maxi. (B) ChargeSwitch® Pro Filter
Plasmid Kits are compatible with the new EveryPrep™ Universal Vacuum
Manifold (K2111-01).
time prior to elution.
Speed of Protocol (minutes)
The new ChargeSwitch® Pro Filter kits have been shown to
purify plasmid DNA with transfection efficiencies equivalent to
15
CST Pro Filter
EndoFree S & P
Miniprep
55
EndoFree Q
120
any anion exchange plasmid kits. ChargeSwitch® Pro Filter kits are
20
ready to go, right out of the box, with all plastics and reagents
Midiprep
25
is no need to add these components to the buffers. The new
ChargeSwitch® Pro Filter kits also provide the high level of bind-
to other kits on the market.
The EveryPrep™ Universal Vacuum Manifold is designed for
120
CST Pro Filter
EndoFree S & P
Maxiprep
ucts. Each kit provides the highest plasmid DNA yield compared
55
EndoFree Q
provided. The buffers do not require salts or ethanol, so there
ing capacity and yield that you expect from our plasmid prod-
CST Pro Filter
EndoFree S & P
55
EndoFree Q
0
20
120
40
60
80
100
120
140
Figure 14. Fastest protocol time. The protocols for the ChargeSwitch® Pro Filter
Plasmid Kits (CST Pro Filter) were compared with competitor endo-free
protocols. The data shows that the entire CST Pro Filter protocol can be
completed in 15–25 min compared to 55–120 min for the competitors’ endotoxin-free protocols.
rapid manual purification of nucleic acids. It offers the ability to
directly elute from the manifold using the new ChargeSwitch®
unit. The manifold reduces sample handling to a minimum by
Pro Filter Plasmid Mini, Midi and Maxi Kits and also many other
allowing direct parallel and simultaneous processing of up to 96
competitors’ vacuum-assisted spin column preps and filter plates.
samples using collection plates, deep-well blocks, or microtubes.
The EveryPrep™ Universal Vacuum Manifold replaces and con-
The use of a vacuum eliminates the need for time-consuming
solidates all previous manifolds into one space -saving benchtop
centrifugation.
Downstream procedures
Ordering information
Product
Quantity
Cat. No.
PureLink™ Quick Plasmid Miniprep Kit
50 preps
250 preps
K2100-10
K2100-11
PureLink™ HiPure Plasmid Filter Maxiprep Kit
10 preps
25 preps
K2100-16
K2100-17
ChargeSwitch® Pro Filter Plasmid Maxiprep Kit
10 preps
25 preps
CS31106
CS31107
EveryPrep™ Universal Vacuum Manifold
1 each
K2111-01
23
www.invitrogen.com
Cloning Tools
After the cloning reaction: electrophoresis
SYBR® Safe DNA Gel Stain
Gel electrophoresis is commonly used to confirm that your clone
→
has the right insert in the proper orientation (if applicable). Here
we discuss several products that help you validate your cloning
Avoid the hazards of UV exposure and get 5-fold greater
sensitivity compared to ethidium bromide
→
results.
Offers nonhazardous waste status and Clean Water Act
compliance
→
Reduces waste storage and disposal costs
E-Gel® EX Precast Agarose Gel Electrophoresis
→
Dual-use gels for both DNA and RNA
SYBR® Safe DNA Gel Stain, available as a ready-to-use solution or
→
5x more sensitive than ethidium bromide
a 10,000X concentrate, can be used like ethidium bromide, either
→
Easy access to gel with openable cassette
in the gel during the electrophoresis run or as a post-run stain.
→
Complete separation in 10 min or less
There is no need to destain before viewing and documenting
The E-Gel® EX precast agarose gel electrophoresis system is a self-
your results. DNA stained with SYBR® Safe stain can be detected
contained, complete system that includes agarose, your choice
using a visible-light transilluminator such as the Safe Imager™ 2.0
of incorporated DNA gel stain, electrodes, and patented ion-
Blue-Light Transilluminator, a standard UV transilluminator, or a
exchange matrices contained inside a disposable, UV-transparent
laser-based scanner.
cassette. With the E-Gel® EX System, you don’t need to pour gels,
make buffer, or even have a power supply. E-Gel® EX precast gels
Safe Imager™ 2.0 Blue-Light Transilluminator
are stable at room temperature and always ready to go—just load
The Safe Imager™ 2.0 Blue-Light Transilluminator is designed for
your samples and run (Figure 15). You’ll have results typically in as
optimal detection of SYBR® dyes such as SYBR® Safe DNA Gel Stain
few as 7 minutes.
(Figure 16).
Step 1. Snap in E-Gel®
EX Pre-Cast Gel.
Step 2. Load samples
and markers.
Figure 15. Using the E-Gel® EX System is as easy as 1-2-3.
24
Step 3. Run with preset or personalized
programs.
Figure 16. Safe Imager™ 2.0 Blue-Light Transilluminator.
TrackIt™ DNA ladders
→
Ready-to-use format—no thawing, diluting, and adding
tracking dye
→
Easy-to-see runs—two tracking dyes run outside the
sample limits during electrophoresis
→
TrackIt™
1 Kb Plus DNA Ladder
TrackIt™
100 bp DNA Ladder
TrackIt™
50 bp DNA Ladder
0.9 μg/lane;
0.9% agarose gel stained with
ethidium bromide.
Cat. No. 10488-085
3 μg/lane;
1.5% agarose gel stained with
ethidium bromide.
Cat. No. 10488-058
0.5 μg/lane;
2% agarose gel stained with
ethidium bromide.
Cat. No. 10488-043
bp –
12,000 –
bp –
5,000 –
Room temperature stable—store on your benchtop for
convenient access
bp –
2,072 –
1,500 –
800 –
2,000 –
600 –
1,650 –
450 –
600 –
TrackIt™ DNA Ladders are offered ready to use. Simply transfer
1,000 –
850 –
TrackIt™ DNA Ladder from the vial to the gel. The two track-
650 –
250 –
500 –
200 –
ing dyes serve as visual markers for following migration during
350 –
300 –
400 –
150 –
electrophoresis and indicate when maximum separation of the
DNA fragments has been achieved. Additionally, visualization of
DNA bands will not be obscured by the tracking dyes because
300 –
100 –
200 –
100 –
100 –
50 –
they run outside the limits of most DNA samples. Three TrackIt™
Figure 17. Three TrackIt™ DNA Ladders.
Ladders are shown in Figure 17. For a complete list of DNA ladders,
visit www.invitrogen.com.
Ordering information
Product
Quantity
Cat. No.
1% E-Gel® EX Starter Kit
1
G6511ST
2% E-Gel® EX Starter Kit
1
G6512ST
1.2% E-Gel® with SYBR® Safe Starter Pak**
1
G6206-01
2% E-Gel® with SYBR® Safe Starter Pak**
1
G6206-02
UltraPure™ Agarose
500 g
16500500
UltraPure™ Agarose 1000
100 g
16550100
UltraPure™ Low Melting Point Agarose
50 g
16520050
UltraPure™ 10X TBE Buffer
10 L
15581044
SYBR® Safe DNA Gel Stain in DMSO (10,000X concentrate)
400 μL
S33102
1L
S33100
80 gels
400 gels
15510-100
15510-500
G6600
Safe Imager™ 2.0 Blue-Light Transilluminator (includes amber filter and viewing glasses)
1
TrackIt™ 1 Kb Plus DNA Ladder
100 apps
10488-085
TrackIt™ 100 bp DNA Ladder
100 apps
10488-058
TrackIt™ 50 bp DNA Ladder
100 apps
10488-043
Downstream procedures
SYBR® Safe DNA Gel Stain in 0.5X TBE (ready-to-use)
UltraPure™ Agarose/SYBR® Safe DNA Gel Stain Combo
** Additional sizes, agarose percentages, and well numbers available. Visit www.invitrogen.com/egels for a complete list.
For the highest-purity reagents to pour your own gels, check out UltraPure™ Agarose products at www.invitrogen.com/ultrapure.
25
www.invitrogen.com
Cloning Tools
Custom DNA oligos
Why do oligos sometimes require purification?
From simple to complex, we have your bases covered
Oligos are made using a DNA synthesizer, a computer-controlled
→
The world’s highest capacity, resulting in prompt, reliable
reagent delivery system. DNA is synthesized in the 3’➝5’ direc-
delivery
tion, and each base addition is accomplished through a series of
Computer-controlled synthesis system eliminates manual
chemical reactions.
→
data transfer and re-entry data, allowing for accurate QC
→
But no chemical reaction is 100% efficient; during DNA syn-
and shipping
thesis, the maximum coupling efficiency obtainable is normally
Rigorous quality control and validation of suppliers and raw
around 99%. Therefore, approximately 1% of the growing nucleo-
materials includes 100% in-process trityl monitoring in real
tide chains fail to couple to a base in each round. In the case of
time and post-synthesis QC by capillary electrophoresis
a 30-mer oligo synthesis, the final product would also contain
29-mer failures, 28-mer failures, etc. For an oligo of this length,
Invitrogen™ Custom DNA oligos are synthetic oligonucleotides
the percentage of full-length oligo is estimated to be between
made according to your specifications and can be used in a vari-
74% and 54%, assuming a 99% or 98% reaction efficiency. The
ety of applications, from PCR and sequencing to probes for gene
percentage of full-length oligo produced decreases as the length
detection. Custom oligos are offered with standard deoxynucleo-
of the oligo increases.
tides, modified bases, and 5´- and 3´-modified nucleotides. Avail-
The failure sequences inherent in the oligo synthesis process
able modifications include fluorescent dyes, enzyme conjugates,
can compete with the full-length product in some applications;
and S-oligos for antisense studies. Invitrogen offers five standard
therefore, to help ensure success in downstream experiments, it
synthesis scales and four purity options (Table 10).
may be necessary to purify the full-length oligo away from the
failure sequences.
Table 10. Benefits of Custom DNA oligo purification methods.
Purification method
Description
Benefit
Desalt
Oligos are processed through normal phase chromatography
columns which removes salts but not failure sequences
A salt-free DNA solution, ready to use; suitable for many PCR
and sequencing applications without further purification
Cartridge
Based on reversed-phase chromatography; removes failure
sequences from the completed synthesis
Provides full-length sequences needed in some applications
HPLC
Reversed-phase high-performance liquid chromatography
(HPLC) removes failure sequences or unincorporated label the
same way as cartridge purification
Guarantees highly purified primer required in some applications (≥85% full-length)
PAGE
Method used to differentiate full-length product from failure
sequences based on size and conformation
Provides the highest percentage of full-length oligos (≥85%)
required for certain demanding applications such as mutagenesis or adapter production
For easy ordering and up-to-date information on Invitrogen™ custom DNA oligos, visit us at www.invitrogen.com/oligos.
26
Custom services
Let the experts do the cloning for you
in bacterial, mammalian, yeast, lentiviral, adenoviral, baculoviral,
Outsource your cloning with our Custom Services
Department
and cell-free expression systems by an LR or BP recombination
reaction.
In addition to our cloning products, we offer custom cloning services
in the following areas: custom gene synthesis, custom ORF cloning;
Custom TOPO® Cloning Adaptation Services
TOPO® Cloning Adaptation Services; DNA fragment production,
If you have a favorite cloning vector or a vector that you wish to use
cloning, and subcloning; cloning PCR products; Gateway® vector
in a specific assay, let our TOPO® Cloning experts TOPO® adapt it for
conversion; Gateway® entry clone cross into destination vectors (L x
you. Now any vector you choose can be adapted for TOPO® Clon-
R crosses); site-directed mutagenesis; custom cDNA library construc-
ing PCR products in 5 minutes at high efficiencies. Our scientists will
tion; and normalization and standardization.
modify your favorite vector by covalently attaching the topoisomer-
For complete information on any of these services, visit www.
invitrogen.com/customservices.
ase I enzyme and preparing the ends for either blunt or TA Cloning®
methods. If you choose, we can also prepare the vector for directional
cloning of PCR products. We’ll functionally test the vector in a TOPO®
Custom Gene Synthesis
Cloning reaction and then send it back to your laboratory for use in
Require synthetic genes for creating challenging constructs and pro-
your HTP cloning. You’ll save time and enhance your research. Your
tein expression studies? Think about the amount of time you spend
TOPO® Cloning-adapted vector is supplied in bulk for a minimum of
at the bench using PCR, mutagenesis and direct cloning to isolate
500 reactions, so it’s HTP compatible. In addition, you’ll receive high-
and prepare your DNA of interest from natural sources, genomic or
efficiency competent cells in bulk or 96-well format for HTP cloning.
cDNA. Gene synthesis outperforms traditional cloning by providing
You won’t have to gather additional reagents to complete your clon-
you fast turnaround times and guaranteed sequence accuracy. Con-
ing projects, saving even more time.
struct design and optimization services are also available in order to
fine tune the expression of your gene(s) of interest. More information
Gateway® Vector Conversion
can be found at www.invitrogen.com/genesynthesis.
The Gateway® Vector Conversion Service includes the following steps:
→
Gateway® vector conversion. Subclone a Gateway® cassette
ORF Cloning Services
into your vector of interest to create a Gateway® destination
The ORF Cloning Services offer a number of options, including:
vector.
→
ORF cloning from a template. PCR cloning from a template
Validation methods. Gateway® LR reaction to show successful
provided by the client or an Invitrogen™ clone collection,
recombination, and sequencing to confirm the cassette is in
cloning into a Gateway® Donor or TOPO® vector, and then full-
frame with a tag if one is present.
length sequencing.
→
→
ORF cloning from a library. PCR cloning from an Invitrogen™
→
Delivery. Converted vector as glycerol stocks, vector maps, and
sequence reports.
cDNA library into a Gateway® Donor or TOPO® vector and then
→
end-read sequencing.
More information
Transfer of ORFs into Gateway® expression vectors. ORFs can
To learn more about these or any of the custom services, visit us online
be transferred to a broad range of Gateway® expression vectors
at www.invitrogen.com/customservices.
27
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www.invitrogen.com
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