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View additional product information for GeneArt™ Site-Directed Mutagenesis System - FAQs (A13282)
15 product FAQs found
We do not offer the DNA methylase from GeneArt Site-Directed Mutagenesis kits as a separate product.
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.
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.
The concentration is 32 mM.
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.
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.
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.
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.
We recommend using AccuPrime Pfx DNA Polymerase with the GeneArt Site-Directed Mutagenesis 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.
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.
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.
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.
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
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