geneart® seamless cloning and assembly kit
DESCRIPTION
The GeneArt® Seamless Cloning and Assembly Kit enables the simultaneous and directional cloning of 1 to 4 PCR fragments, consisting of any sequence, into any linearized vector, in a single 30-minute room temperature reaction. The kit contains everything required for the assembly of DNA fragments, and their transformation into E. coli for selection and growth of recombinant vectors. • Speed and Ease — Clone up to 4 DNA fragments, with sequence of your choice, simultaneously in a single vector (up to 13 Kb); no restriction digestion, ligation or recombination sites required • Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind • Vector Flexibility — Use our linear vector or a vector of your choice • Free Tools — Design DNA oligos and more with our free web-based interface that walks you step-by-step through your project • Diverse Applications — Streamline many synthetic biology and molecular biology techniques through the rapid combination, addition, deletion, or exchange of DNA segments For cloning more than 4 DNA fragments, final molecules larger than 110 Kb, or for using pre-existing DNA elements too long to be amplified by PCR; please consider the GeneArt® High-Order Genetic Assembly System (cat# A13285). Simple and Fast Clone Creation GeneArt® Seamless Cloning is a simple, two step process, consisting of a tube based assembly reaction followed by transformation into One Shot® Chemically Competent TOP10 E. coli. The kit uses the properties of a proprietary enzymatic mix to assemble DNA fragments with shared terminal end homology without leaving any extra sequences or scars behind (seamless). A portion of the assembly reaction is then transformed into the provided competent TOP10 cells, yielding clones ready for analysis the next day. The required 15-base pair end-homology can be easily engineered by PCR-amplification with custom DNA oligos. Cloning Efficiency, Flexibility, and Precision With the GeneArt® Seamless Cloning and Assembly Kit the main factors effecting cloning efficiency are the size of the DNA elements (100 bp to 5 Kb), the total size of the final molecule (≤ 13 Kb), and the quality and specificity of the fragment. The terminal end of the PCR fragments (A-overhangs or blunt), does not affect the cloning efficiency. Typical cloning efficiencies for different numbers of fragments cloned into pUC19L are the following: • 90% for a single 5 Kb DNA element • 70% for 4 fragments of 1 Kb each • 40% for 4 DNA fragments of 2 Kb each For more information visit: https://products.invitrogen.com/ivgn/product/A13288?CID=cloningkit-SS-12312TRANSCRIPT
GeneArt® Seamless Cloning and Assembly SystemThe GeneArt® Seamless Cloning and Assembly System enables cloning of up to 4 DNA fragments simultaneously into virtually any linearized vector,
typically in 30 minutes, without extra DNA sequences, restriction endonucleases, or ligation.
Product Quantity Cat. No.
GeneArt® Seamless Cloning and Assembly Kit 20 reactions A13288
GeneArt® Linear pUC19L Vector for Seamless Cloning 20 reactions A13289
GeneArt® High-Order Genetic Assembly SystemThe GeneArt® High-Order Genetic Assembly System is a highly efficient, vector-independent system for the simultaneous and seamless assembly of
up to 10 DNA fragments (preexisting or synthetic) in vivo, totaling up to 110 kb in construct size.
Product Quantity Cat. No.
GeneArt® High-Order Genetic Assembly System 10 reactions A13285
GeneArt® High-Order Genetic Assembly System (with Yeast Growth Medium) 10 reactions/2 L medium A13286
GeneArt® High-Order Linear pYES1L Vector 10 reactions A13287
CSM Medium for Mav203 Yeast Cells 2 L A13292
GeneArt® High-Order Vector Conversion Cassette 10 reactions A13291
GeneArt® Site-Directed Mutagenesis SystemThe GeneArt® Site-Directed Mutagenesis System is a simple and highly efficient method for in vitro site-directed mutagenesis. The system can gener-
ate base substitutions, deletions, or insertions in DNA plasmids from any source, without specialized vectors, host strains, or restriction sites.
Product Quantity Cat. No.
GeneArt® Site-Directed Mutagenesis System 16 reactions A13282
Component Size Quantity
10X Enzyme Mix 45 μL/tube 1 tube
5X Enzyme Buffer 90 μL/tube 1 tube
Linear pUC19L Vector (50 ng/μL), 4 control reactions 8 μL/tube 1 tube
Control insert (50 ng/μL) 5 μL/tube 1 tube
Component Size Quantity
One Shot® TOP10 Chemically Competent E. coli 50 μL/tube 21 tubes
pUC19 Control (10 pg/μL) 10 μL/tube 1 tube
S.O.C Medium 6 mL/bottle 1 bottle
Component Size
DNA Methylase (4 units/μL) 20 μL
200X SAM (S-adenosine methionine) 10 μL
10X Enhancer 100 μL
0.5 M EDTA 500 μL
Component Size
pUC19WHITE Control Plasmid (20 ng/μL) 100 ng
Control Primer Mix (10 μM) 25 μL
PCR Water 1.8 mL
5X Reaction Buffer 90 μL
Component Size
10X Enzyme Mix 45 μL
One Shot® MAX Efficiency® DH5α™ T1R 1 box
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GeneArt® Genetic Assembly Tools
geNeTic AssembLy
• Speed—clone up to 4 DNA fragments (up to 13 kb total size) simultaneously in a single tube, at room temperature, typically in 30 minutes
• Flexibility—use our linear vector or any vector of your choice
• Precision and efficiency—no extra sequences; just clone what you want where you want it
• Simplicity—online DNA oligo Designer designs oligos and assembles DNA molecules in silico
The GeneArt® Seamless Cloning and Assembly System uses a proprietary enzyme mix to recognize and precisely assemble DNA fragments sharing a 15–base pair end homology. End homology is created by PCR amplification using primers designed to generate overlap between adjacent DNA fragments to be assembled.
The online DNA Oligo Designer (www.invitrogen.com/designdnaassembly) guides users through experimental design, including oligo design and ordering.
GeneArt® Seamless Cloning and Assembly SystemThe geneArt® seamless cloning and Assembly system enables cloning of up to 4 DNA fragments simultaneously into virtually any linearized vector, typically in 30 minutes, without extra DNA sequences, restriction endonucleases, or ligation.
Assembledcircular construct
One Shot® TOP10ChemicallyCompetent E. coli
6–8 µL of the reaction mix
Incubate 20–30 minutes on iceHeat shock for 30 secondsAdd 250 µL SOCRecover for 1 hour at 37°C
10–50 µL of the transformation mix
Plate on selective LB platesIncubate overnight at 37°C
Pick 4–10 colonies
Isolate the assembled construct from 4–10 positive coloniesAnalyze by restriction digest and/or sequencing
DNA fragments
Incubate 30 minutes atroom temperature
Design PCR oligos using DNA Oligo Designer, and PCR-amplify the DNA fragmentsto add the required homologyLinear vector
XX
X
DNA fragments and linear vector recombine
GeneArt® Seamless Cloning and Assembly System workflow
Figure 1. The GeneArt® kit demonstrates higher cloning efficiency than product “I” when more than two inserts are cloned simultaneously into a linear vector. The two kits were compared for cloning efficiency using the same vector and inserts in four cloning reactions. 1: one insert and vector; 2: two inserts and vector; 3: three inserts and vector; 4: four inserts and vector.
Number of inserts
Clon
ing
effic
ienc
y (%
)
GeneArt® Seamless Cloning and Assembly SystemProduct ‘I’
1 kbp fragments, 15 bp overlaps,AccuPrime™ Pfx polymerase
120
100
80
60
40
20
01 2 3 4
• Speed—clone 10 or more DNA fragments simultaneously into a single vector (up to 110 kb total); assemble existing DNA fragments without restriction digestion or pcR amplification
• Flexibility—use our linear vector or a vector of your choice; oligonucleotide stitching feature allows end-editing and reuse of preexisting DNA fragments without the need for reamplification
• Precision and efficiency—no extra sequences; just clone what you want where you want it
• Simplicity—online DNA oligo Designer walks you through your project step by step; design your DNA oligos and assemble your DNA molecule in silico (www.invitrogen.com/designdnaassembly)
GeneArt® High-Order Genetic Assembly SystemThe geneArt® High-order genetic Assembly system is a highly efficient, vector-independent system for the simultaneous and seamless assembly of up to 10 DNA fragments (preexisting or synthetic) in vivo, totaling up to 110 kb in construct size.
Figure 3. Cloning efficiency of the GeneArt® High-Order Genetic Assembly System with increasing numbers of 10 kb PCR fragments. Even as 100 kb of PCR fragments are assembled, the cloning efficiency remains over 65%, proving the system is a reliable solution for assembly of complex DNA molecules.
Number of 10 kb DNA fragments
Clon
ing
effic
ienc
y (%
)
100
80
60
40
20
01 3 10
x x x x x x
x x x x x x
Fragmentsize
1 x 1 kb
2 x 5 kb
[3 x 5 kb] + [2 x 0.5 kb][3 x 5 kb] + [2 x 0.5 kb]
2 x 5 kb
2 x 5 kb
2 x 5 kb
Oligosize
60-mer
80-mer
60-mer80-mer
80-mer10 bp insertion
80-mer20 bp insertion
80-mer12 bp insertion
Cloningefficiency
94%
75%
37%75%
63%
50%
87%
x x
x x
x x
x x
x x
x x
x x
x x
x x
x x
x x
x x
x x x x x x
x x
x x x x x x
x x x x x xv
v
Figure 2. Oligonucleotide stitching. (A) Oligonucleotide stitching enables joining of unrelated DNA fragments. (B) Oligonucleotide stitching can be used for junction editing.
A
B
The GeneArt® High-Order Genetic Assembly System relies on yeast’s ability to take up and recombine DNA fragments with high efficiency via transformation-associated recombination, greatly reducing in vitro handling of DNA and eliminating the need for restriction digestion and ligation.
In cases where DNA fragments do not share end homology (i.e., existing DNA fragments not created by PCR), they can be “stitched” together using recombinant linkers that provide sequence overlaps to promote recombinatorial joining of unrelated DNA fragments (Figure 2A).
In addition, oligonucleotide stitch-ing allows editing of the fragment junctions to generate deletions and insertions (Figure 2B). This feature also allows the reuse of existing DNA fragments or elements obtained by restriction enzyme digestion without the need to reamplify them by PCR.
• Control—insert, delete, or change up to 12 nucleotides in plasmids up to 14.5 kb
• Speed—entire protocol, including transformation, is completed typically in less than 3 hours (using a 3 kb plasmid)
• Precision and efficiency—mutagenesis efficiency over 90% (using a 3 kb plasmid)
The GeneArt® Site-Directed Mutagenesis System utilizes mutagenic oligonucleotide primers to generate mutations. The mutagen-esis protocol is streamlined by combining DNA methylation and amplification steps into a single reaction, eliminating post-mutagenesis diges-tion and purification steps.
The GeneArt® Site-Directed Mutagenesis System delivers superior mutagenesis per-formance with a wide range of vector sizes. A comparative analysis against competitor “Q” reveals the advantage of using the GeneArt® Site-Directed Mutagenesis System for a single–base pair mutation (Figure 4).
As the size of the template plasmid increases, the superior mutagenesis efficiency of the GeneArt® system is clearly demonstrated.The flexibility, efficiency, and speed make the GeneArt® Site-Directed Mutagenesis System the ideal choice for your routine and complex site-directed mutagenesis needs.
GeneArt® Site-Directed Mutagenesis SystemThe geneArt® site-Directed mutagenesis system is a simple and highly efficient method for in vitro site-directed mutagenesis. The system can generate base substitutions, deletions, or insertions in DNA plasmids from any source, without specialized vectors, host strains, or restriction sites.
Plasmid size (kbp)
Mut
agen
esis
effi
cien
cy (%
)
GeneArt® Site-Directed Mutagenesis System
Competitor ‘Q’
100
90
80
70
60
50 2.8 5.9 8.9
Methylatedplasmid
xxx
x
x
x
x
xx
x
Mutatedplasmid
x
xx
Methylation
Mutagenesis
In vitrorecombinationreaction
Transformation
Step 2: Recombination reaction
Add 1 µL 0.5 M EDTA to stop the reactionUse 2 µL of recombinant reaction sample for transformation
Incubate on ice for 15 minutesHeat-shock for 30 secondsAdd 250 µL SOC mediumRecover for 1 hour at 37°C
Step 3: Transformation
Step 1: Methylation
2. Perform the in vitro recombination reaction, which enhances the colony output 3x to 10x.
3. Transform the sample into DH5α™-T1® competent E. coli. The host cell circularizes the linear mutated DNA, and McrBC endonuclease in the host cell digests the methylated template DNA, leaving only unmethylated mutated product.
1. Methylate plasmid DNA and amplify the plasmid in a mutagenesis reaction with two overlapping primers containing the target mutation.
Steps 1–3:
Plasmid template PCR ReagentsMethyl Transferase Reagents
PCR sample10X enzyme mixReaction buffer
Figure 4. GeneArt® Site-Directed Mutagenesis System vs. competitor “Q”.
GeneArt® Site-Directed Mutagenesis System protocol