Hi All,
I am looking to add 3 nucleotides to a position in my DNA sequence which won't disrupt the coding region but introduce a restriction site into it. Is there any way I can add 3 different base pairs to my sequence manually?
Thank you all in advance.
Cheers
Hello,
Why did you not proceed by PCR? you can add whatever you want in your primer.
Best regards
Assuming you have cloned your coding sequence in a plasmid, you can insert a sequence of choice using primer-site-directed mutagenesis. The method is most often described for point mutations, but it works pretty well for insertional mutagenesis.
Thank you both for your answers. I am thinking of using QuickChange kit which uses primers to make site-directed mutagenesis but i was just thinking if the kit is better for 3 base pair changes in single go.
Definitely, you can insert the 3 bp by one round of mutagenesis. We have done larger insertions successfully.
Does quickchange allow that. The primer designing software they provided does not have insertions in more than 1 sites at the same time
Then I recommend to use the standard design principle ("by hand"): To the mutant sequence (3 nt) add 20 flanking nucleotides upstream and downstream; longer flanks if GC content is
- Badges
- Science method