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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.
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.
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.
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.
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