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Do-it-yourself Gene Synthesis Kit

I cannot cut out my gene of interest using Xba1. Why is this?

We have a variety of strains that are used in production, such as Top10 or DH5α™. Routinely, we grow them in dam+ strains. Therefore, you may be seeing inhibition because Xba1 is sensitive to dam methylation.

I don’t think my sequence is accurate. What should I do?

Please send an email to geneartsupport@thermofisher.com explaining what is wrong. We will also need the project ID and construct ID, which we will forward to our QC department for further investigation.

I am not getting a PCR product using your GeneArt® Do-It-Yourself Gene Synthesis Kit. What should I do?

Overdigestion with CorrectASE™ enzyme can lead to degradation of the DNA template.

Ensure that the reaction does not go longer than 60 mins. Also, ensure that the reaction is kept on ice until the PCR step or else the reaction will be prone to overdigestion by the CorrectASE™ enzyme.

I’m confused with the primer concentration to use for the Do It Yourself Gene Synthesis Kit. Can you please clarify?

The protocol states that oligonucleotide stocks should be prepared at a final concentration of 100 μM in 1X TE buffer. The next line indicates the addition of 5 μL of each 10 μM primer together. According to R&D, the manual was written this way because our R&D typically brings up the lyophilized oligos to a 100 μM stock concentration (due to the volume of the tube). You do, however, want to use a 0.15 μM pool. Therefore, you can either dilute the stock to 10 μM or dilute the primer pool 1:10.

GeneArt® Site-Directed Mutagenesis Kits

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.
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’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.

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