Molecular cloning

workflow solutions
Clone | Transform | Purify | Analyze
 Clone Transform

Purify Analyze

2
 Contents
Advance your cloning workflow Clone
Cloning solutions overview 5
The molecular cloning workflow requires many Cloning solutions 6
steps to obtain the perfect clone for downstream
applications. Advance your cloning workflow at Transform
every step—clone, transform, purify, and analyze to Competent cells 11
successfully produce a recombinant clone quickly. Medium- and high-throughput transformation 13

This handbook is intended to guide you by providing Purify

technical information and clear choices across the Plasmid purification solutions 15
cloning workflow with solutions designed for speed Solutions for molecular applications 16
Solutions for transfection applications 17
and simplicity.

With all of our cloning solutions, you can find what fits Analyze
and get the results you need to move closer to the Sample analysis solutions 19
Sample isolation solutions 21
next step in your discovery.

Choose your simple, but powerful, cloning Resources

solutions and all necessary resources at Frequently asked questions 23
Mobile apps 23
thermofisher.com/pcrandcloning.
Ordering information 24

3
Cloning

Molecular cloning relies on recombinant DNA technologies to insert a DNA sequence of interest into a vector to
generate a large number of copies of the recombinant vector.
Cloning has traditionally required restriction enzymes and a DNA ligase to form a new recombinant vector.
However, recent cloning advancements, such as Invitrogen™ TOPO™ cloning, ligation-independent cloning, and
gene synthesis, provide more efficient workflow solutions.

4
 Cloning

Cloning solutions overview

For more than 25 years, we have provided superior tools for DNA cloning, continually improving upon
traditional technologies and developing new ones. From restriction enzymes to complete cloning kits, we
offer a comprehensive portfolio of tools and reagents that will help you save resources while obtaining
high-quality cloned DNA to accelerate your next discovery.

Do you intend to build multiple

fragments or genes without any
extraneous sequences?

Yes No

Invitrogen™ GeneArt™ Gibson Assembly® cloning kits Is this for routine cloning and subcloning?
GeneArt Gibson Assembly HiFi Cloning Kit,
chemically competent cells
Yes No
GeneArt Gibson Assembly HiFi Master Mix
GeneArt Gibson Assembly EX Cloning Kit,
chemically competent cells
Are sequence and restriction mapping Does your research require easy shuttling of
GeneArt Gibson Assembly EX Master Mix information for the source material available? DNA among a variety of host–vector systems?

Yes No Yes No

Thermo Scientific™ FastDigest™ Invitrogen™ TOPO ™ Cloning and TOPO ™ Invitrogen™ Gateway™ cloning systems with Invitrogen™ GeneArt™ Strings™ DNA
restrictionand modifying enzymes TA Cloning™ kits Clonase™ enzyme mixes Fragments and other custom services
TOPO TA Cloning Kit for Subcloning, Gateway BP Clonase II Enzyme Mix Find out more about GeneArt Strings DNA
FastDigest BamHI FastDigest KpnI
without competent cells Gateway LR Clonase II Enzyme Mix Fragments and fully cloned genes at
FastDigest BcuI FastDigest NotI
Zero Blunt TOPO PCR Cloning Kit, MultiSite Gateway Pro Plus thermofisher.com/geneart
FastDigest BshTI FastDigest SaII without competent cells
PCR Cloning System with Gateway Technology,
FastDigest DpnI FastDigest XbaI pENTR/D‐TOPO Cloning Kit, with One Shot with pDONR 221 and OmniMAX 2 Competent Cells
FastDigest EcoRI FastDigest XhoI TOP10 Chemically Competent E. coli
TOPO XL‐2 Complete PCR Cloning Kit, with One Shot
Type IIs: OmniMAX 2 T1R Chemically Competent E. coli
FastDigest Esp3I (BsmBI)
FastDigest BpiI (BbsI)
FastDigest Eco31I (BsaI)

Find more cloning tips and troubleshooting advice at

thermofisher.com/cloningeducation and thermofisher.com/cloningsupport

5
Cloning solutions
TOPO cloning Gateway cloning system
Invitrogen™ TOPO™ PCR cloning technology was developed to help improve To shuttle genes of interest between vectors, the Gateway cloning system
cloning efficiency, simplify protocol setup, and accommodate a wide range offers site-specific, recombination-based cloning. With this easy-to-use
of PCR insert sizes. TOPO technology enables inserts with compatible ends choice for cloning in multiple expression systems, the insert’s proper
to be readily joined to the vector in 5 minutes, without the need for additional orientation and reading frame are maintained during shuttling using the
ligation steps. Gateway vectors.

• High-fidelity, blunt-end TOPO cloning—designed for cloning PCR Find out more at thermofisher.com/gateway
products amplified with high-fidelity polymerases; includes the ccdB gene
for positive selection
ccdB

• TOPO TA cloning—for fast, effective, direct cloning of Taq polymerase– attL1 attL2 attR1 attR2
amplified PCR products with overhangs or sticky ends Entry
clone + Destination
vector

• TOPO long-fragment cloning—for easy, highly efficient cloning of

extra-long PCR products using the linearized, topoisomerase I-activated KanR AmpR

pCR™-XL-2-TOPO vector
BP Clonase LR Clonase

Add 1 µL of PCR Perform transformation

Perform PCR with Taq or reaction to 1 µL of Incubate for 5 minutes at with provided competent ccdB
proofreading polymerase TOPO cloning vector room temperature E. coli strain

PCR product A attB1 attB2 attP1 attP2

or PCR product

+
A

or
CACC
PCR product Expression Donor
TOPO
clone vector
TOPO

TOPO
cloning
vector AmpR KanR

The Gateway cloning reactions. This scheme shows the four types of plasmids and enzyme
The three steps of TOPO PCR cloning technology. mixes involved in Gateway cloning reactions. Red arrows represent the fragment of interest.
Adapted from Katzen F (2007) Expert Opin Drug Discov 2(4):571–589.

Find out more at thermofisher.com/topocloning

TOPO cloning selection guide

Quickly find your TOPO cloning kit with our new interactive selection
tool. Search by application, vector, or desired competent cells at
thermofisher.com/topoguide

6
 Cloning

Found naturally in bacteria, restriction enzymes recognize and cleave specific DNA sequences, resulting
Double-
in sticky ends (5ʹ­or 3ʹ protruding ends) or blunt ends, which enable DNA inserts to be cloned into stranded
DNA
vectors with compatible ends. Star activity, buffer compatibility, and incomplete digestion are some
common hurdles in restriction digestion.
Restriction enzyme

FastDigest restriction enzymes

To simplify cloning, we offer FastDigest enzymes, an advanced line of restriction enzymes that share
buffer compatibility with downstream modifying enzymes. Benefits include:
Sticky ends
• Complete digestion in • No sequential digestions and
5–15 minutes buffer changes
• Double and multiple digestions • 176 unique specificities
in a universal buffer for any • Direct loading of the reaction
combination of enzymes mixture on gels
One effect of this offset cleavage is that the still-intact unless the two fragments have been intentionally designed One effect of this offset cleavage is that the still-intact
recognition sequence remains with one fragment while the to be compatible. With such upfront design (done by recognition sequence remains with one fragment while th
Find out more at thermofisher.com/fastdigest
other fragment loses the recognition sequence. A second choosing or creating type IIs–compatible cloning vectors other fragment loses the recognition sequence. A secon
effect is that Type IIs restriction
compatible enzymes
A B while the precise distance of the cleavage site and generating cloning fragments via gene effect is that while the precise distance of the cleavage s
from the recognition site is enzyme-dependent, as is the
Plasmid DNA digested with FastDigest enzymes Plasmid DNA digested with NEB enzymes
Asynthesis
specific group
or PCR of restriction
primer enzymes,
design), workflows can be called type
from the IIs endonucleases,
recognition site is enzyme-dependent, as is the
type of cleavage (blunt, 3´ protruding, or 5´ protruding), the simplified and cloning products can be scarless, whichtype of cleavage (blunt, 3´ protruding, or 5´ protruding), th
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 cleave DNA outside of their recognition sequences. In combination with DNA
sequence at the cleavage site is typically irrelevant; once can be an important consideration when dealing with sequence at the cleavage site is typically irrelevant; once
the enzyme binds to its recognition site, it will cleave any ligase, typeframes.
open reading IIs restriction enzymes
It is also possible are utilized
to simultaneously to drive the insertion of one or
the enzyme binds to its recognition site, it will cleave any
DNA sequence the appropriate distance away. The result several
assembleDNA fragments
multiple inserts in a into a recipient
specific order into avector
DNAwithout
sequencethe
the inclusion
appropriate of residual
distance away. The resul
of this mechanism is that two different DNA fragments single vector [4].
restriction enzyme sites and other unwanted DNA sequences at fragment fragments
of this mechanism is that two different DNA
cleaved by a given type IIs restriction enzyme that leaves cleaved by a given type IIs restriction enzyme that leaves
protruding ends will not have compatible overhangs—
junctions (scarless cloning).
protruding ends will not have compatible overhangs—

A B A
Find FastDigest type IIs enzymes at thermofisher.com/fastdigesttypeiis

Type II enzyme Type IIs enzyme Type II enzyme

1: undigested plasmid DNA 6: FastDigest XmaJI 1: undigested plasmid DNA 6: AvrII
2: FastDigest BcuI 7: FastDigest Eco31I 2: SpeI-HF 7: BsaI
3: FastDigest XbaI 8: FastDigest Eco52I 3:
N XbaI
N N N N G A A T T C N 8:
N EaqI-HF
N N N N N N N G A A G A C N N N N N N N N N N N N N N N N N N N G A A T T C N N N N
4: FastDigest NdeI 9: FastDigest BglII 4: NdeI 9: BglI
5: FastDigest SalI 5: SalI-HF
C T T A A G N N N N N C T T C T G N N N N N N N N N N N N N N
N N N N N N N N N
C T T A A G
N N N N N N N N N

Figure 1. Comparison of digestion efficiencies of restriction enzymes. (A) FastDigest

restriction enzymes digest plasmid DNA much more efficiently compared to the (B) NEB enzymes.
In this experiment, using the NEB protocol, 1 μg of plasmid DNA was digested for ~15 minutes. Recognition site and cleavage site of type IIs vs. type II restriction enzymes.
Figure 1. Type II vs. type IIs restriction endonucleases. Representative (A) type II (EcoRI) and (B) type IIs (BpiI) enzymes are shown. The DNA binding Figure 1. Type II vs. type IIs restriction endonucleases. Representa
and DNA cleavage activities of typical type II enzymes are closely tied together in a single protein domain, as is reflected in their cleavage patterns. Type
and IIs
DNA cleavage activities of typical type II enzymes are closely tied to
enzymes bind at one DNA sequence, but a second protein domain cleaves DNA at a specified distance in a sequence-independent manner. 7
enzymes bind at one DNA sequence, but a second protein domain clea
Cloning solutions (cont.)
GeneArt Gibson Assembly cloning kits
GeneArt Gibson Assembly cloning kits allow for simultaneous assembly of
up to 15 large DNA fragments to precisely create very large constructs with DNA fragments Linear vector Recombine DNA fragments
and linear vector
no additional sequences in highly efficient reactions. This cloning method
circumvents the need for multiple rounds of restriction enzyme analysis and
digestion, DNA end repair, dephosphorylation, ligation, enzyme inactivation,
and cleanup, and is a powerful tool in synthetic biology. OneShot TOP10 chemically competent
Assembled circular or ElectroMax DH10B electrocompetent
construct E. coli transformation mix
Benefits of GeneArt Gibson Assembly kits:
• Assembly of up to 15 fragments to build seamless clones
Figure 2. Overview of the Gibson Assembly reaction, which relies on homologous
• Cloning efficiencies up to >95% recombination to assemble adjacent DNA fragments sharing end-terminal homology.
• Choice of complete kits with competent cells or master mixes
Find out more at thermofisher.com/gibsonassembly Gibson Assembly EX master mix and kit:
Invitrogen™ GeneArt™ Gibson Assembly® EX Master Mix and Invitrogen™
Gibson Assembly HiFi kits: GeneArt™ Gibson Assembly® EX Cloning Kit provide high transformation
Invitrogen™ GeneArt™ Gibson Assembly® HiFi kits offer a very cost-effective efficiency for larger assemblies.
and efficient way of assembling smaller numbers of fragments.
Cloning efficiency
Cloning efficiency
6 inserts 8 inserts 10 inserts
1 insert 2 inserts 3 inserts
100%
100%

80%
80%

60%
60%

40%
40%

20%
20%

0%
0% GeneArt Gibson GeneArt seamless
GeneArt Gibson NEBuilder kit In-Fusion kit Assembly EX kit cloning
Assembly HiFi kit (NEB) (Takara)

Figure 1. GeneArt Gibson Assembly HiFi kits provide high cloning efficiency using Figure 3. GeneArt Gibson Assembly EX kits provide high transformation efficiency options
single- to multiple-insert designs. Assembly of 1, 2, or 3 fragments of 1 kb in Invitrogen™ when using larger numbers of inserts. Assembly of 6, 8, and 10 fragments of 0.5 kb in pcDNA
pcDNA™ 3.4 vector using Invitrogen™ TOP10 competent cells. GeneArt Gibson Assembly HiFi kits 3.4 vector transformed into Invitrogen™ One Shot™ TOP10 competent cells. GeneArt Gibson
are the most cost-effective and time-saving method for building large assemblies, particularly Assembly HiFi and EX technologies are available as master mixes or complete kits with chemically
when used with GeneArt Strings DNA Fragments or 100% sequence-verified Invitrogen™ GeneArt™ competent or electrocompetent cells, so you can have the flexibility of using your –20°C or –80°C
Gene Synthesis. freezer.

8
 Cloning

Cloning with synthetic DNA

If you lack GeneArt
the time to generate
Gene and clone insert DNA, including optimization and
Synthesis
troubleshooting, our synthetic DNA fragments
A reliable and cost-effective method and cloning service
for obtaining mightDNA
customized be right for you. G
A G
A
A
G
G

GeneArt Strings DNAwith

constructs Fragments and GeneArt
100% sequence Gene Synthesis
accuracy, offer genes
GeneArt Gene analogous
Synthesis offers: to www
5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘

optimized, error-free PCR products.

Bioinformatics

Fragments
A C G C
• Synthetic, ready-to-transfect genes

DNA Fragments
Day 1 Oligo synthesis Day 2 Day 3 Day
GeneArt Strings DNAtoFragments
• The ability clone into many popular Invitrogen™ vectors or your own Ordering until overnight GeneAssembler Cloning Sequencin
3:00 p.m. (CET) process quality co
A time-saving alternative
custom to PCR, GeneArt Strings DNA Fragments are available in lengths
vector

Strings DNA
A G
A

up to 3 kb•and
Fully cloned, 100%with
are compatible any downstream cloning method
for of choice, providing: Oligonucleotide
G A G
G

sequence-verified genes ready www

5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘

synthesis

Strings
• Synthetic, downstream
ready-to-useapplications
DNA fragments A C G C A

• Free optimization of a gene with Invitrogen™ GeneArt™ GeneOptimizer™ Day 1 Oligo synthesis Day 2 Day 3 Day 4 D

GeneArt
• DNA with your specified ends to facilitate the cloning method of choice Ordering until overnight GeneAssembler Cloning Sequencing and Rea

GeneArt
software for maximum protein expression
3:00 p.m. (CET) G
process quality control ship
• No starting
FindDNA required
A A

out more at thermofisher.com/genesynthesis G A G

G

Synthesis
5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘
www

Gene assembly

Gene Synthesis
• Free optimization of gene with Invitrogen™ GeneArt™ GeneOptimizer™ software for A C G C A
GeneArt Strings DNA Fragments Day 1 Oligo synthesis Day 2 Day 3 Day 4 Day 5
maximum protein expression
A time-saving alternative to PCR, GeneArt Strings DNA Fragments are Ordering until overnight GeneAssembler Cloning Sequencing and Ready for

GeneArt Gene
3:00 p.m. (CET) process quality control shipment
• Option ofavailable
Strings in
DNAlengths up towith
Libraries 3 kbmixed,
and are compatible
randomized with any downstream
nucleotides using full
cloning method of choice, providing:
IUPAC code

GeneArt
A G
A
G A G

Cloning
G
5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘
www
• Synthetic,
Find out more ready-to-use DNA fragments
at thermofisher.com/strings A C G C A

• DNA with your specified ends to facilitate the cloning method of choice Day 1 Oligo synthesis Day 2 Day 3 Day 4 Day 5
Ordering until overnight GeneAssembler Cloning Sequencing and Ready for
GeneArt Gene Synthesis
• No starting DNA required 3:00 p.m. (CET) process quality control shipment
A reliable and cost-effective method for obtaining customized DNA constructs with 100%
sequence •accuracy,
Free optimization of the Synthesis
gene with offers:
GeneArt GeneOptimizer software for G

GeneArt Gene
A A
G A G
G

Sequencing
5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘
www
maximum protein expression
• Synthetic, ready-to-transfect
• Option of Invitrogengenes
™
GeneArt™ Strings™ DNA Libraries with mixed, A C G C A

Day 1 Oligo synthesis Day 2 Day 3 Day 4 Day 5

randomized
• Cloning into nucleotides
several available using
vectors full IUPAC
(custom codes
options available)
Ordering until overnight GeneAssembler Cloning Sequencing and Ready for
3:00 p.m. (CET) process quality control shipment
• 100% sequence-verified
Find out more at and ready for downstream applications
thermofisher.com/strings
www
G
A G
A
A
G
G

5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘ 5‘
Final
• No starting DNA required
A C G C A
documentation
• Free optimization of gene with GeneOptimizer software Day
for1maximumOligo synthesis
protein Day 2
expression Day 3 Day 4 Day 5
Ordering until overnight GeneAssembler Cloning Sequencing and Ready for
3:00 p.m. (CET) process quality control shipment
Find out more at thermofisher.com/genesynthesis
9
42
Transform
After the DNA fragment is cloned into a vector,
competent bacteria are transformed with it to propagate
sufficient quantities of the cloned DNA for downstream
experiments. The choice of competent cells for
transformation depends upon the transformation
methods, strain genotypes, plasmid characteristics, and
desired applications.

Find more transformation tips and troubleshooting advice at

thermofisher.com/compcells and thermofisher.com/compcells-education
10
 Transform

Competent cells
Choosing competent cells based on the application
We offer a broad portfolio of chemically competent and electrocompetent cells in a wide range of
transformation efficiencies, formats, and strains. Use the decision tree below to select the best cells for
your specific application area.

Is this for routine cloning and subcloning?

Yes No

Routine cloning Specialized cloning Protein production

Yes No
Is high transformation ef ficiency needed?
Is this for gDNA or library production?
Yes No Yes No
Protein production
Contact technical support
High transformation Standard ef ficiency Is this for unstable inser ts? One Shot BL21 Star (DE3) thermofisher.com/contact
gDNA or library production
efficiency (cloning) (propagation) High mRNA stability for
One Shot OmniMAX 2 T1R Yes No increased protein expression
One Shot OmniMAX 2 T1R Subcloning Efficiency
(>20 kb) One Shot BL21(DE3)
(5 x 109 cfu/µg) DH5α (1 x 106 cfu/µg)
ElectroMAX DH10B Is this for methylation- High-level expression
One Shot TOP10 (>20 kb) sensitive plasmid DNA? of recombinant proteins
(1 x 109 cfu/µg)
ElectroMAX DH10B T1R Unstable inserts One Shot BL21(DE3) pLysE
One Shot DH10B T1R (>20 kb) Low basal expression of
MAX Efficiency Stbl2
(1 x 109 cfu/µg) No T7 promoter genes
MegaX DH10B T1R One Shot Stbl3 Yes
MAX Efficiency DH5α Electrocomp (>100 kb) (lentiviral vectors) One Shot BL21(DE3) pLysS
(1 x 109 cfu/µg) ElectroMAX Stbl4 Low basal expression, faster
Library Efficiency DH5α* Is this for Invitrogen induction of T7 promoter genes
(1 x 109 cfu/µg) Methylation-sensitive Gateway destination One Shot BL21-AI
One Shot Mach1 T1R vector propagation? Best for expression of
(1 x 109 cfu/µg) One Shot INV110
toxic proteins

Yes No

GatewayGateway destination
destination Contact technical support
vector thermofisher.com/contact
Electrocompetent cells.
One Shot ccdB
Survival 2 T1R
* Not for methylated or genomic DNA.

Find out more at thermofisher.com/compcells

11
Competent cells (cont.)
Chemically competent cells Electrocompetent cells
Chemically competent cells are treated with calcium chloride to facilitate Electrocompetent cells are used in the electroporation process. Electrical
attachment of the plasmid DNA to the cell membrane. The competent cells pulses create pores that allow genetic material to permeate the bacterial
are heated in a water bath—this opens the pores of the cell membrane, membrane. The Invitrogen™ electrocompetent cell portfolio offers a variety of
allowing entry of the plasmid. Chemically competent cells are the best E. coli cells to reliably clone your DNA with high efficiency.
solution for general cloning and subcloning applications.
ElectroMAX DH10B cells
One Shot TOP10 cells These electrocompetent E. coli cells offer the highest transformation
Chemically competent One Shot TOP10 E. coli cells are ideal for high- efficiencies of >1 x 1010 cfu/μg plasmid DNA. Invitrogen™ ElectroMAX™ DH10B™
efficiency cloning and plasmid propagation and are provided at a cells are ideal for applications requiring high transformation efficiencies, such
transformation efficiency of 1 x 109 cfu/μg plasmid DNA. They allow stable as with cDNA or gDNA library construction.
replication of high copy number plasmids and are the same competent cells
that come with many of our cloning kits.

Heat shock
Incubation (e.g., 42°C, 30 sec) Recovery

Chemical or
electrocompetent cell
Electric shock
(e.g., 15k V/cm. 5 μsec pulses)

Chemical and electrocompetent transformation.

Find out more at thermofisher.com/compcells

12
 Transform

Medium- and high-throughput transformation

Performing bacterial transformations one by one can be very time-consuming and create a bottleneck in
your experimental workflow. There are times when medium- and high-throughput transformation options
are desired. Invitrogen™ MultiShot™ chemically competent cells provide three flexible product formats to
meet your throughput needs.

Find out more at thermofisher.com/multishot

StripWell format

• Medium-throughput option

• Twelve 8-tube strips

• Suitable for 1–96 transformations

• Five E. coli strains available

FlexPlate format

• High-throughput option

• 96-well plate separates into twelve 8-well segments

• Manual and automated platform transformations

• Six E. coli strains available

96-well plate
Did you know?
• Highest-throughput option
Invitrogen™ competent cells can be
• Five 96-well plates provided in custom configurations
per your request. Large and custom
• Available with the One Shot TOP10 strain
volumes, as well as multiple formats,
• Stable replication of high copy number plasmids are at your fingertips. Simply email us at
[email protected]

13
Purify
Once the bacterial cells are transformed with the vector,
it is critical to recover plasmid DNA using plasmid
purification products that are optimized to provide
maximum yield, purity, and integrity.

Find more plasmid purification tips and troubleshooting resources at thermofisher.com/plasmid

and thermofisher.com/plasmidsupport
14
 Purify

Plasmid purification solutions

Plasmid purification is a basic technique necessary for generating recombinant clones. Thermo Scientific™
GeneJET™ and Invitrogen™ PureLink™ plasmid purification kits have been designed and developed with
your experimental specifications, plasmid purity, and throughput demands in mind. Use the decision tree
below to select the right plasmid purification kit based on the intended application.

What is the intended use of the isolated plasmid?

Molecular applications like cloning,

PCR, and sequencing

Yes No

PureLink Quick kits GeneJET kits

Endotoxin level: >10.0 EU/µg Endotoxin level: >10.0 EU/μg

Prep size: mini Prep size: mini, midi, maxi
Protocol duration: 30–45 min Protocol duration: 15–60 min Transfection of robust cell lines like HEK and CHO
Yield: up to 40 µg Yield: 0.02–1 mg
Culture volume: 1–5 mL Culture volume: 1–200 mL
Technology: Silica membrane Technology: Silica membrane

Yes No

Transfection of sensitive cells like primary cell cultures,

Gibco™ Expi Expression System, and in vivo use
PureLink HiPure kits PureLink Fast Low-Endotoxin kits

Endotoxin level: <1 EU/μg Endotoxin level: <1 EU/μg

Yes No
Prep size: mini, midi, maxi, mega, giga Prep size: midi, maxi
Protocol duration: 90–120 min Protocol duration: 15–30 min
Yield: 0.015–14 mg Yield: 0.4–1.5 mg PureLink Expi Endotoxin-Free kits Contact technical support
Culture volume: 1 mL–5 L Culture volume: 30–150 mL thermofisher.com/contact
Endotoxin level: <0.1 EU/μg
Technology: Anion exchange Technology: Advanced silica membrane
Prep size: maxi, mega, giga
Protocol duration: 90–120 min
Yield: 0.4–15 mg
Culture volume: 100 mL–5 L
Technology: Anion exchange

Find out more at thermofisher.com/plasmid

15
Solutions for molecular applications
GeneJET and PureLink kits
Robust purification of plasmid DNA, in the amount and purity required for the downstream application of
interest, is a key step of the cloning workflow. During the construction of plasmid DNA and subsequent
verification (sequencing, enzymatic digestion), small-scale plasmid DNA preparations are generally used
(see the table below). Silica membrane–based columns such as GeneJET and PureLink products are
ideal due to their fast and simple purification workflows.

These kits offer the following benefits:

• High yields of low-throughput, molecular-grade plasmid DNA

• Ideal for basic molecular biology applications, including PCR, sequencing, cloning, and transcription

• Thermo Scientific™ GeneJET™ Plasmid Miniprep, Midiprep, and Maxiprep kits offer the best overall
value and ease of use, and the fastest processing (for conventional pellet method)

• The Invitrogen™ PureLink™ Plasmid Miniprep Kit offers a slightly higher yield and comes more complete
(elution tubes plus optional buffers included) in mini-scale column format

Find out more at thermofisher.com/genejet and thermofisher.com/purelink

Scale considerations for plasmid purification

Parameter Miniprep Midiprep Maxiprep Megaprep Gigaprep
Bacterial culture volume 1–5 mL 10–50 mL 100–500 mL 500 mL–2.5 L 2.5–5 L
Plasmid yield Up to 40 µg Up to 300 µg Up to 1 mg Up to 4 mg Up to 15 mg

16
 Purify

Solutions for transfection applications

PureLink kits
For experiments involving cells and animal models, endotoxin levels are one of a scientist’s primary
concerns. Endotoxins can induce immune responses, resulting in suboptimal transfection and
toxicity in many cell lines, and negatively influence protein expression in sensitive cells. To enable
successful in vitro and in vivo studies, Invitrogen™ PureLink™ HiPure, Fast Low-Endotoxin, and Expi
Endotoxin-Free kits are ideal for applications that require high purity of plasmids and low-to-“zero”
levels of endotoxins.

Find out more at thermofisher.com/plasmid

Maxi Maxi

1.5 1.5
<0.05 EU/µg <0.05 EU/µg

1.2 1.2
<0.05 EU/µg PureLink Fast Low-Endotoxin kit <0.05 EU/µg PureLink Expi kit
Supplier Q Plasmid Plus kit Supplier Q EndoFree kit
0.9 0.9
Yield (mg)

Yield (mg)
0.6 0.6

0.3 0.3

0 0
1 2 1 2
Plasmid Plasmid

Figure 5. Higher yields of low-endotoxin, advanced transfection-quality plasmid DNA Figure 6. Higher yields of endotoxin-free, advanced transfection-quality plasmid DNA
obtained with a PureLink Fast Low-Endotoxin purification kit than with a comparable obtained with a PureLink Expi purification kit than with a comparable kit from another
kit from another supplier. Two high-copy plasmids with different backbones were purified supplier. Two high-copy plasmids with different backbones were purified using the PureLink
using the PureLink Fast Low-Endotoxin and Supplier Q maxiprep kits as described in the Expi Endotoxin-Free and Supplier Q maxiprep kits as described in the product manuals. Plasmid
product manuals. Plasmid yields are shown. Endotoxin values (EU/μg) were measured using an yields are shown. Endotoxin values (EU/μg) were measured using the EndoSafe-PTS test and are
Endosafe™-PTS instrument (Charles River Laboratories) and are provided only for the PureLink provided only for the PureLink Expi preparations.
Fast Low-Endotoxin preparations.

17
Analyze

Choosing the right tools for your cloning analysis can significantly improve and accelerate results, enabling you
to address downstream applications sooner. Explore our precast analysis options, do-it-yourself reagents, and
isolation kits to find the right workflow solution for your cloning analysis needs.

Find more nucleic acid analysis tips and troubleshooting resources at

thermofisher.com/na-electrophoresis-education
18
 Analyze

Sample analysis solutions

Identifying the appropriate gel type and gel concentration for your analysis is an essential step that will
streamline the separation of nucleic acids. Find out more about our convenient reagents for sample
analysis using agarose gel electrophoresis, including pour-your-own Invitrogen™ UltraPure™ agarose
reagents and hassle-free, precast Invitrogen™ E-Gel™ agarose gels in this section.

Does your cloning workflow require quality-control analysis and rapid results?

Yes No

Precast gel electrophoresis Pour-your-own gel electrophoresis

Are you analyzing 22 samples or less? Are you performing fragment gel extraction?

Yes No Yes No

E-Gel precast agarose gels for E-Gel precast agarose gels for Pour-your-own gel analysis and Pour-your-own gel
fast DNA analysis high-throughput analysis fragment isolation reagents analysis reagents

E‐Gel Power Snap Electrophoresis System E‐Gel E‐Base Electrophoresis UltraPure LMT Agarose UltraPure Agarose
E‐Gel EX Precast Agarose Cassettes System (High Throughput) UltraPure Water UltraPure Water
E‐Gel DNA Ladders E‐Gel 48/96 Precast Agarose Cassettes SYBR Safe DNA Gel Stain SYBR Gold DNA Gel Stain
E‐Gel DNA Ladders TrackIt DNA Ladders TrackIt DNA Ladders
PureLink Quick Gel Extraction Kit GeneJET PCR Purification Kit
Are you extracting fragments from a gel? UltraPure Buffers UltraPure Buffers
Owl EasyCast B2 Gel Owl EasyCast B2 Gel
Yes No Electrophoresis Systems Electrophoresis Systems

E-Gel system fragment

DNA cleanup reagents
isolation reagents
E‐Gel Power Snap System GeneJET PCR Purification Kit
E‐Gel CloneWell II Precast Agarose Cassettes
E-Gel Double Comb Agarose Gels
E‐Gel DNA Ladders

Find out more at thermofisher.com/egel

19
Sample analysis solutions (cont.)
E-Gel precast gels Electrophoresis reagents
Using precast agarose gels can simplify the nucleic acid electrophoresis For pour-your-own agarose gels, choosing
workflow. E-Gel precast gels are self-contained and ready for use, with the high-quality agarose, optimized DNA ladders,
agarose and the DNA stain packaged in a disposable cassette. The use of and improved DNA stains can help you achieve
in-gel Invitrogen™ SYBR™ Safe DNA Gel Stain and a blue-light transilluminator optimal electrophoresis results.
minimizes DNA damage and improves cloning efficiency compared to
conventional methods. There are no gels to pour, buffers to make, staining
or destaining steps to perform, or gel boxes to assemble. Just load your
samples and start the run. UltraPure reagents for electrophoresis
Invitrogen™ UltraPure™ reagents are specifically formulated to meet your
nucleic acid analysis and purification needs. UltraPure agarose and reagents
are made from highly pure biochemicals for maximum reliability and
superior performance.

Find out more at thermofisher.com/ultrapure

DNA stains
Detection of nucleic acid samples in gels can be improved using fluorescent
dyes that are safer and more sensitive than ethidium bromide. SYBR Safe and
Invitrogen™ SYBR™ Gold Nucleic Acid Gel stains provide greater safety and
sensitivity with lower background fluorescence than the conventional ethidium
bromide stain.

Sample analysis in three simple steps—load, run, and analyze. Find out more at thermofisher.com/stains

Find out more at thermofisher.com/egel DNA ladders

Invitrogen™ DNA Ladders are available for a wide variety of size ranges,
E-Gel Power Snap Electrophoresis System applications, and formats. SYBR Safe stain has lower background
To help reduce workflow time, the Invitrogen™ E-Gel™ Power Snap fluorescence than the conventional ethidium bromide stain. They accurately
Electrophoresis System integrates rapid, real-time nucleic acid analysis with estimate size and mass of double-stranded, single-stranded, or supercoiled
high-resolution image capture. DNA fragments.

Find out more at thermofisher.com/powersnap Find out more at thermofisher.com/ladders

20
 Analyze

Sample isolation solutions

Whether isolating a specific DNA fragment from complex PCR mixtures or
recovering bands from agarose gels, we have solutions that will meet your
needs. Isolate DNA fragments that are ready for a variety of downstream
applications like sequencing, PCR, transcription, cloning, and labeling.

1 2 3 4 5 6 7 8 9 10 11

DNA load = 25 ng/lane

Lane 1: 1 kb Plus DNA Ladder
Lane 2: 100 bp amplicon, unpurified
Lane 3: 100 bp amplicon, purified
Lane 4: 400 bp amplicon, unpurified
Lane 5: 400 bp amplicon, purified
Lane 6: 965 bp amplicon, unpurified
Lane 7: 965 bp amplicon, purified
Lane 8: 2 kb amplicon, unpurified
Lane 9: 2 kb amplicon, purified
Lane 10: 5.4 kb amplicon, unpurified
Lane 11: 5.4 kb amplicon, purified

Amplification of DNA isolated using the Invitrogen™ PureLink™ Quick Gel Extraction Kit.
PCR amplicons varying in size from 100 bp to 5.4 kb were prepared using recombinant
Invitrogen™ Taq DNA Polymerase. A portion of each PCR reaction was run on a 1% Invitrogen™
UltraPure™ Agarose gel (data not shown), and amplicon bands were excised and extracted using
the PureLink Quick Gel Extraction Kit. The Invitrogen™ 1 Kb Plus DNA Ladder was used in this gel.

Invitrogen kits for DNA cleanup

Product Protocol time (min) DNA cleanup application Format Elution volume (µL) Quantity
50 preps
GeneJET PCR Purification Kit 5 PCR cleanup Silica spin or vacuum column 50
250 preps
50 preps
PureLink Quick Gel Extraction Kit <30 Gel extraction Silica spin or vacuum column 30–100
250 preps

Find out more at thermofisher.com/cleanup

21
Resources
A comprehensive portfolio of educational resources,
frequently asked questions, and mobile apps are now
available to help you reach new heights in your research.

Find more cloning tips and troubleshooting advice at

thermofisher.com/cloningeducation and thermofisher.com/cloningsupport

22
 Resources

Frequently asked questions Mobile apps

Below are some common questions and answers to help you start or
troubleshoot your molecular biology experiments. Instrument Connect—remote monitoring
With Instrument Connect—the remote monitoring app
Clone powered by Connect, our cloud-based platform—you can
What are some of the prerequisites for TOPO cloning? stay connected to any cloud-enabled instrument, including
Please consider the following before you do TOPO cloning: the Applied Biosystems™ ProFlex™, SimpliAmp™, and
MiniAmp™ PCR instruments.
• TOPO cloning cannot ligate DNA with a 5ʹ phosphate group.

• TOPO cloning will decrease in efficiency inversely with the size of the insert
(>3 kb) unless you use the TOPO XL cloning kit.

• TOPO vectors contain different antibiotic resistance markers, which should

be considered before purchase. Purify
What growth conditions do you recommend for E. coli for
Are GeneArt Gibson Assembly kits available with isolating plasmid DNA using your plasmid isolation kits?
electrocompetent cells? We typically recommend growing E. coli up to A600 = 1–1.5 (~1 x 109 cells/mL)
Yes, both the GeneArt Gibson Assembly HiFi and EX configurations in LB broth.
are available with electrocompetent cells. Go to thermofisher.com/
gibsonassembly to see the full list of available products. Can I elute my plasmid from PureLink columns with water instead
of TE?
What is the main difference between GeneArt Strings DNA Fragments For any silica columns, elution with water is generally possible. However, a
and GeneArt Gene Synthesis? buffer is preferred for stability and accuracy of absorbance readings, as pure
GeneArt Strings DNA Fragments are custom-made, uncloned, water can have a very low pH (4–5).
double-stranded linear DNA fragments. GeneArt Gene Synthesis is a
service offered for chemical synthesis, cloning, and sequence verification of Analyze
genetic sequences. I want to pour my own gels. Which agarose should I use?
The UltraPure Agarose is standard, melting‐point agarose designed
Transform for routine separation and analysis of DNA and RNA fragments in the
Can you explain the significant differences between the TOP10, 500–23,000 nt range. Invitrogen™ UltraPure™ Agarose-1000 is a specialized
DH5α, and Mach1 strains that you have for the cloning reactions? agarose that provides higher resolution of PCR fragments and other short
DH5α cells are commonly used for routine cloning, but are mcr/mrr + and DNA fragments. We also offer an Invitrogen™ UltraPure™ Low Melting Point
therefore not recommended for genomic cloning. The TOP10 competent Agarose, which is ideal for resolving and isolating DNA fragments from 10 to
cells, on the other hand, are mcr/mrr —, and therefore are a good choice for 1,000 bp with a low melting temperature of 65°C or less.
routine cloning and can be used for cloning of methylated DNA, such as
eukaryotic genomic DNA. Our Mach1 strain is the fastest-growing cloning
strain that is T1 phage-resistant. 23
Ordering information
Product Quantity Cat. No.

Clone
FastDigest BamHI 800 reactions FD0054
FastDigest BcuI 50 reactions FD1254
FastDigest BshTI 20 reactions FD1464
FastDigest DpnI 100 reactions FD1704
FastDigest EcoRI 800 reactions FD0274
FastDigest KpnI 300 reactions FD0524
FastDigest NotI 50 reactions FD0594
FastDigest SaII 200 reactions FD0644
FastDigest XbaI 300 reactions FD0684
FastDigest XhoI 400 reactions FD0694
FastDigest Esp3I (BsmBI) (type IIs) 20 reactions FD0454
FastDigest BpiI (BbsI) (type IIs) 20 reactions FD1014
FastDigest Eco31I (BsaI) (type IIs) 100 reactions FD0294
TOPO TA Cloning Kit for Subcloning, without competent cells 10 reactions 451641
Zero Blunt TOPO PCR Cloning Kit, without competent cells 10 reactions 451245
Zero Blunt TOPO PCR Cloning Kit for Sequencing, with One Shot TOP10 Chemically Competent E. coli 25 reactions K287520
pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli 20 reactions K240020
TOPO XL-2 Complete PCR Cloning Kit, with One Shot OmniMAX 2 T1R Chemically Competent E. coli 20 reactions K8050-20
TOPO TA Cloning Kit for Subcloning, without competent cells 10 reactions 451641
Gateway LR Clonase II Enzyme Mix 20 reactions 11791020
MultiSite Gateway Pro Plus 20 reactions 12537100
MultiShot FlexPlate Stbl3 Chemically Competent E. coli 1 plate C7381201
PCR Cloning System with Gateway Technology with pDONR 221 and OmniMAX 2 Competent Cells 20 reactions 12535029
GeneArt Gibson Assembly HiFi Cloning Kit, chemically competent cells 10 reactions A46624
GeneArt Gibson Assembly HiFi Master Mix 10 reactions A46627
GeneArt Gibson Assembly EX Cloning Kit, chemically competent cells 10 reactions A46633
GeneArt Gibson Assembly EX Master Mix 10 reactions A46635
GeneArt Gene Synthesis thermofisher.com/genesynthesis

24
Product Quantity Cat. No.

Transform
One Shot TOP10 Chemically Competent E. coli 20 reactions C404003
MultiShot FlexPlate TOP10 Chemically Competent E. coli 1 plate C4081201
MAX Efficiency DH5α Competent Cells 200 µL 18258012
MultiShot FlexPlate DH5α T1R Chemically Competent E. coli 1 plate C4481201
ElectroMAX DH10B Cells 100 µL 18290015
MAX Efficiency Stbl2 Competent Cells 5 x 200 µL 10268019
One Shot Stbl3 Chemically Competent E. coli 20 x 50 µL C737303
MultiShot FlexPlate Stbl3 Chemically Competent E. coli 1 plate C7381201
Purify
10 preps A31217
PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit 25 preps A31231
4 preps A33073
PureLink Expi Endotoxin-Free Mega Plasmid Purification Kit 4 preps A31232
PureLink Expi Endotoxin-Free Giga Plasmid Purification Kit 2 preps A31233
PureLink Expi Endotoxin-Free Buffer Set 1 set A33074
25 preps A35892
PureLink Fast Low-Endotoxin Midi Plasmid Purification Kit
50 preps A36227
25 preps A35895
PureLink Fast Low-Endotoxin Maxi Plasmid Purification Kit
50 preps A36228
25 preps K210002
PureLink HiPure Plasmid Miniprep Kit
100 preps K210003
25 preps K210004
PureLink HiPure Plasmid Midiprep Kit
100 preps K210005
10 preps K210006
PureLink HiPure Plasmid Maxiprep Kit
25 preps K210007
PureLink HiPure Expi Plasmid Megaprep Kit 4 preps K210008XP
PureLink HiPure Expi Plasmid Gigaprep Kit 2 preps K210009XP
50 preps K210010
PureLink Quick Plasmid Miniprep Kit (molecular grade)
250 preps K210011
25 preps K0481
GeneJET Plasmid Midiprep Kit
100 preps K0482
10 preps K0491
GeneJET Plasmid Maxiprep Kit 25 preps K0492
50 preps K0502
GeneJET Plasmid Miniprep Kit 250 preps K0503

25
Ordering information (cont.)
Product Quantity Cat. No.

Analyze
UltraPure Ethidium Bromide 10 mL 15585011
UltraPure Agarose 100 g 16500100
UltraPure DNase/RNase-Free Distilled Water 10 x 500 mL 10977023
UltraPure TAE Buffer 10X 4L 15558026
TrackIt 1 Kb Plus DNA Ladder 50 µg 10488085
SYBR Safe DNA Gel Stain 400 μL S33102
E-Gel Agarose Gels EX, 1% 10 gels G401001
E-Gel Agarose Gels EX, 2% 10 gels G401002
E-Gel Agarose Gels EX, 4% 10 gels G401004
E-Gel Agarose Gels with SYBR Safe DNA Gel Stain, 1% 10 gels A42100
E-Gel Agarose Gels with SYBR Safe DNA Gel Stain, 2% 10 gels A42135
E-Gel Agarose Gels with SYBR Safe DNA Gel Stain, 4% 10 gels A42136
E-Gel EX Double Comb Agarose Gels, 1% 10 gels A42345
E-Gel EX Double Comb Agarose Gels, 2% 10 gels A42346
E-Gel Double Comb Agarose Gels with SYBR Safe DNA Gel Stain, 1% 10 gels A42347
E-Gel Double Comb Agarose Gels with SYBR Safe DNA Gel Stain, 2% 10 gels A42348
E-Gel CloneWell II Agarose Gels with SYBR Safe, 0.8% 10 gels G661818
E-Gel SizeSelect II Agarose Gels, 2% 10 gels G661012
E-Gel NGS 0.8% Agarose Gels 10 gels A25798
E-Gel Go! Agarose Gels, 1% 10 gels G441001
E-Gel Go! Agarose Gels, 2% 10 gels G441002
E-Gel 48 Agarose Gels with SYBR Safe DNA Gel Stain, 1% 8 gels G820801
E-Gel 48 Agarose Gels with SYBR Safe DNA Gel Stain, 2% 8 gels G820802
E-Gel 48 Agarose Gels with SYBR Safe DNA Gel Stain, 4% 8 gels G820804
E-Gel 96 Agarose Gels with SYBR Safe DNA Gel Stain, 1% 8 gels G720801
E-Gel 96 Agarose Gels with SYBR Safe DNA Gel Stain, 2% 8 gels G720802
PureLink Quick Gel Extraction Kit 50 preps K210012
GeneJET PCR Purification Kit 50 preps K0701

Find out more at thermofisher.com/pcrandcloning

For Research Use Only. Not for use in diagnostic procedures. © 2019–2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher
Scientific and its subsidiaries unless otherwise specified. Endosafe is a trademark of Charles River Laboratories, Inc. Gibson Assembly is a registered trademark of SGI-DNA, Inc. NEBuilder is a
trademark of New England Biolabs, Inc. In-Fusion is a trademark of Takara Bio USA, Inc. COL34351 1121