If I design primers end to end for making the transformation cassette using 500-700bp of the gene segment, I doubt if the those sequences (upstream and downstream) synthesize some structural component of the protein whole expression I am aiming to silence.
Please let me know if the primers for the recombination cassette should be designed from promoter or the gene itself.
Michael J. Benedik
If your goal is to knockout the function of a gene then even an internal deletion of that gene should suffice. But there may be situations where you want to abolish all transcription in which case deleting part of the promoter might be useful. But for most situations deleting the entire gene or some internal segment of it should suffice.
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