i wanted to delete a strech of 120 amino acids present between N and C terminal of my protein. is i possible to remove such a long stretch by quick change method? any specific consideration for primer design? i mean the length of primer and the stretch of amino acids to be taken for designing primer?
120 amino acids will be about 360 bp, too long for a deletion. The best way I think is introducing two restriction enzyme sites but for one restriction enzyme at the beginning and end of the part you plan to delete by site directed mutagenesis. Digest your DNA with that one restriction enzyme and let the linearized DNA strand to self-ligate upon ligation. You can insert a triplet of palindromic to the C-terminal of the (will be) N-terminal part and the N-terminal of the (will be) -terminal part.
Hi. Why not try whole plasmid PCR (sometimes referred to as inverse PCR)? We do it all the time.
I assume your gene is cloned into a plasmid. Simply design two primers (please refer to attached file for description), and whole plasmid amplify your plasmid vector. I would like to include a restriction enzyme site to re-join the two ends or you could order phosphorylated primers to do a blunt end ligation. You need to use high fidelity polymerase such as pfu or Q5 or kappa Hifi etc.
yes my ene is cloned in a plasmid and i want to delete an internal region of the gene.thank u so much
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