Hi All
I am designing a primer for 3 nucleotide deletions in my gene . These nucleotides are located toward the end of my gene near the tag on my plasmid . I have tried multiple primers and non of them seems to work. Any advice ...
thank you
Did you use the NEB software (https://nebasechanger.neb.com/ ) to design your primers ?
Hi Sumaiah,
Is this the kit you are using, or are you trying to do your SDM another way?
https://international.neb.com/products/e0554-q5-site-directed-mutagenesis-kit#Product%20Information
More information about how long your primers are, their sequence, which gene it is or the actual mutations you're trying to incorporate would be useful, so that people can give you specific advice. Also, what are the problems you are having (what is it that's not working)? Do you get colonies, but none of them have the plasmid with your desired mutations, or is the problem something else? I usually do SDM the 'old-school' way without using a kit, but I recently struggled with a few mutations and the NEB Q5 SDM kit solved my problems.
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