Invitrogen™ GeneArt™ Site-Directed Mutagenesis System

Catalog No.	Quantity
A13282	16 Reactions

Catalog No. A13282 Supplier Invitrogen™ Supplier No. A13282

Description

Make substitutions, deletions, or insertions of up to 12 nucleotides in plasmids up to 14kb in vitro

The GeneArt™ Site-Directed Mutagenesis System provides a state-of-the-art, simple, convenient, and highly efficient means to generate mutations on a target construct in vitro in less than three hours. This system replaces the popular GeneTailor™ Site-Directed Mutagenesis System, and has been completely redesigned to be at the leading edge of commercial site-directed mutagenesis products currently available on the market. This product brings our mutagenesis technology to the next level. Note: A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.

• Powerful – Make substitutions, deletions, or insertions of up to 12 nucleotides in plasmids up to 14kb
• Efficient – Enables you to obtain your desired mutant the first time; over 90% correct mutants for a 3kb plasmid
• Fast – Have your mutated plasmid DNA in less then 3 hours with the simple, minimal handling protocol
• Versatile – Use plasmids of many sizes, and DNA isolated from any source, with no need for specialized vectors, host strains, or restriction sites

Simple Creation of Desired Mutants
Creating mutants with the GeneAr™ Site-Directed Mutagenesis System relies on the inherent properties of DNA methylase, high fidelity DNA polymerase, recombination enzymes, and the native McrBC endonuclease of E. coli. Simply incorporate the mutation or mutations (up to 12 nucleotides), that you want into primers, and after PCR, recombination, and transformation you get vectors with only the mutations you desired with up to 90% efficiency. The template vector that you add mutations to can be isolated from any source and up to 14Kb in size. For creating complete constructs, or for joining large pieces of unrelated DNA see our GeneArt™ Seamless Cloning and Assembly Kit (cat# A13288) or our GeneArt™ High-Order Genetic Assembly System (cat#A13286).

Optimized Mutagenesis Protocol
The GeneArt™ Site-Directed Mutagenesis System has been optimized for efficiency and simplicity. The DNA methylation and amplification steps are combined into a single reaction with no need for an in vitro DpnI digestion step. After methylation and amplification, a 10 minute in vitro recombination reaction of the amplified PCR products increases the colony output by 3 to 10 fold; resulting in a higher mutagenesis efficiency. A final transformation of the mutated DNA into DH5α™ E. coli cells digests any methylated parental DNA, leaving behind only the intact unmethylated mutagenesis reaction product. No purification steps or additional digestions are needed. Individual colonies can be selected the following day to verify mutations.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

• Bacterial or Yeast Strain: DH5α
• Cell Type: Chemically Competent E. coli
• Content And Storage: Each GeneArt™ Site-Directed Mutagenesis System includes enough reagents for 16 reactions and a control: DNA methylase, S-adenosylmethionine, enzyme mix and buffer, enhancer, 0.5M EDTA, sterile water, pUC19WHITE mutagenesis control, and One Shot™ MAX Efficiency DH5α™-T1R chemically competent cells.
  
  A PCR enzyme is not included with the system and must be purchased separately. The recommended enzyme for this kit is AccuPrime™ Pfx DNA Polymerase.
  
  Store the -80°C Module and the E.coli competent cells at -80°C.
  
  Store all other components at -20°C.
  
  Guaranteed stable for 6 months when properly stored.
• Efficiency: 90%
• For Use With (Application): Seamless Cloning & Genetic Assembly
• Mutagenesis Capability: Single-Site Mutagenesis
• Mutagenized Region Length (up to): 12 nucleotides
• Mutation Type: Deletions, Insertions, Substitutions
• Plasmid: Plasmids up to 14 kb
• Product Line: GeneArt
• Product Type: Site-Directed Mutagenesis System
• Quantity: 16 Reactions

Frequently Asked Questions (FAQs)

Is the DNA methylase from GeneArt Site-Directed Mutagenesis kits available separately?

We do not offer the DNA methylase from GeneArt Site-Directed Mutagenesis kits as a separate product.

How does PCR with two overlapping primers result in a final product with homology at both ends?

During the first cycle of PCR on the circular template, the polymerase will generate two linear products that are able to anneal and undergo primer extension in the next cycle. PCR products that incorporate the mutation at both ends will accumulate in subsequent cycles. After PCR, the recombination reaction results in circularization of the PCR product which enhances the mutagenesis efficiency.

How do I design my primers for my mutation using the GeneArt Site-Directed Mutagenesis System? How large of a mutation site can I have and where should the mutation be located in the primer? How long should the primer be?

The primer should be 30-45 nucleotides in length, with the mutation centrally located. The mutation should be flanked by at least 15-20 nucleotides on either side, depending on the size of the mutation, with the primer length no longer than 45 nucleotides. We recommend using our online GeneArt primer design tool.

What is the concentration of 200X SAM in the GeneArt Site-Directed Mutagenesis System?

The concentration is 32 mM.

What is the difference between the GeneArt Site-Directed Mutagenesis Kit and the older discontinued GeneTailor kit?

The differences are as follows:

- The GeneArt kit combines the methylation and DNA amplification steps, saving 1 hour in the protocol.
- The enzyme mix from the GeneArt Seamless Cloning and Assembly Kit is part of the protocol, boosting the colony output without the need to do many PCR cycles (another time saving step).
- The new kit also contains an enhancer used during the PCR step that increases mutagenesis efficiency for a wide range of plasmid sizes.

What is the difference between the GeneArt Site-Directed Mutagenesis PLUS Kit and the GeneArt Site-Directed Mutagenesis System?

The kits are almost the same, but the PLUS kit contains an improved 2X GeneArt enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites in plasmids up to 14 kb). The PLUS kit can also be used for single-site mutations of up to 25 nucleotides, while the original kit can be used for single substitution/deletion/insertions up to 12 nucleotides in plasmids up to 14 kb. A web tool is available for primer design that can also be used for the original kit.

I'd like to have site-directed mutagenesis performed as a service by GeneArt off of my own plasmid. Can this be done?

Yes. Add “single variant” as a new process, then choose an “upstream service” which is: gene synthesis, already at Geneart, or I will provide this master gene. If the customer is providing their own gene, they will need to fill in the field for the complete sequence of their vector + insert. If it is a plasmid only, they will put the mutagenized area as the “insert” and the rest of the plasmid as the “vector”. The “insert” can only be up to 4kb long.

Do I have to make a fresh dilution of 25X SAM each time I perform my GeneArt Site Directed Mutagenesis procedure?

Yes, 25X SAM is not stable and will lose activity within a few hours. We recommend creating a new dilution of SAM from the 200X SAM supplied in the kit each time you perform a mutagenesis procedure.

What polymerase do you recommend using with the GeneArt Site-Directed Mutagenesis and GeneArt Site-Directed Mutagenesis PLUS systems?

We recommend using AccuPrime Pfx DNA Polymerase with the GeneArt Site-Directed Mutagenesis kits.

What is the difference between the GeneArt Site-Directed Mutagenesis and GeneArt Site-Directed Mutagenesis PLUS kits?

The kits are almost the same, but the PLUS kit contains an improved 2X GeneArt enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites). A web tool was also introduced for primer design, but this can also be used for the original kit.

Do you offer a kit for site-directed mutagenesis?

Yes, we offer three kits for site-directed mutagenesis: GeneArt Site-Directed Mutagenesis System and GeneArt Site-Directed Mutagenesis PLUS kit, Phusion Site-Directed Mutagenesis Kit as well as a custom GeneArt service.

The kits are almost identical, but the PLUS kit contains an improved 2x GeneArt Enzyme mix with protocols optimized for multi-site mutagenesis (up to 3 sites in plasmids up to 14 kb). The PLUS kit can also be used for single-site mutations of up to 25 nucleotides, and the original kit can be used for single substitution/deletion/insertions up to 12 nucleotides in plasmids up to 14 kb. A web tool was also introduced for primer design, but this can also be used for the original kit. Phusion Site-Directed Mutagenesis Kit is a versatile and efficient tool for introducing point mutations, insertions, or deletions in any type of plasmid DNA. With this kit, the entire plasmid is amplified using phosphorylated primers that introduce the desired changes. The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with T4 DNA Ligase. The resulting plasmid can be then transformed into any competent E. coli cells.

After a PCR mutagenesis protocol, how is the plasmid repaired after it is transformed into E. coli?

The detailed mechanism is still not very clear, but involves the E. coli double strand break repair system. The key steps are:

(1) An exonuclease, like RecBCD, removes nucleotides from the ends to generate a single-stranded region.

(2) Strand invasion with homologous regions occurs.

(3) Repair enzymes including nucleases, ligase, and DNA polymerases repair gaps and/or nicks.

I'm getting high background when testing my mutagenesis reaction product. What should I do?

Please see the typical causes and solutions for this problem below:

-Inactive DNA methylase or inactive SAM: Test the activity of the DNA methylase and 25X SAM using the methylation control reaction.
-Too much DNA used: Use no more than 50 ng of DNA per 50 µL of methylation reaction.
-Over-amplification: Reduce PCR cycles to 12 for small plasmids or 15 for intermediate size plasmids.

I'm performing site-directed mutagenesis and am not seeing a visible PCR product. What should I do?

Please see the typical causes and solutions for this problem below:

-Too little DNA: use up to 50 ng DNA per 50 µL reaction.
-Poor quality DNA: purify new plasmid.
-Incorrect DNA polymerase used: We recommend using 1 unit of AccuPrime Pfx DNA Polymerase for amplification.
-PCR conditions were incorrect: Optimize annealing temp and extension time.
-Poor primer design: Use the GeneArt Primer and Construct Design Tool to reduce potential secondary structures or increase primer length

I followed the recommended PCR conditions for my site-directed mutagenesis studies but am not getting a product. What should I do?

Please review the following recommendations:

-What polymerase are you using? We recommend using AccuPrime Pfx DNA polymerase with both mutagenesis kits - use 1 unit of the polymerase for amplification.
-Try optimizing the annealing temperature and extension time. The rule of thumb is 5-10 degrees C below your primer's lowest melting temperature for the annealing temperature, and 30 seconds per 1kb for extension time. You can experiment with different temperatures/times to optimize your reaction.
-Poor primer design could result in no product. We recommend using our tool to reduce potential secondary structures or increase the primer length.
-Check the quality of your starting DNA sample.
-Ensure that you are adding enough DNA to the PCR reaction

For Research Use Only. Not for use in diagnostic procedures.