Hello dear colleagues, i did the site directed mutagenesis of the plasmid with Thermo Scientific Phusion Site-Directed Mutagenesis Kit to introduce a point mutation with phosphorylated primers. When i obtained the resulting plasmid after cloning, i Sanger sequenced it. But the chromatogramm signals interrupts right before the mutagenic primer annealing site (Fig.). What could be the reason for it? Thank you.
Can you attach an original .abi sequence file please?. Did you sequence only in one direction and does the reverse sequence fail at approximately the same position?
To check mutagen, be sure to sequence pair ends and be sure to choose the right company with the right services for the job. Explain to them about the purpose of the work and the desired result before working. I use the services of Kiagene Fanavar Company, which are located at our university. I will send the link and email of the company, they will answer your questions professionally
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email: [email protected]
It is because the sequence may have a G repeat in such scenario, sequencing is often difficult due to formation of hairpin or loop formation or even dGTP depletion. To recover such cases there can be a two options first you can use dGTPBigdye terminator kit v 3.0 or 2nd option is to try for opposite strand sequence so that you can get the mutation site
If the issue is the N's in your sequence, you can annotate the file based on what you see as the peak (this one looks like AGA to me). Sometimes you just get a poor sequence run. I agree with sequencing from both directions. Can you do a digestion check too?
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