I am planning to design Revers primer and add His-tag to it . His-tag sequence is CAT CAT CAT CAT CAT CAT (6 histidin). They are added to Revers primer but :
Should they be added to Revers primer complementary and reversely?
is this sequence correct? : CCCGGGTTA ATG ATG ATG ATG ATG ATG CAGCT
Always consider the directionality!
I suppose your reverse primer sequence above is written from 5' to 3'?
So let's take the His-tag sequence for example (double-stranded DNA):
5'-CAT CAT CAT CAT CAT CAT-3'
3'-GTA GTA GTA GTA GTA GTA-5'
The mRNA produced from this will be:
5'-CAU CAU CAU CAU CAU CAU-3'
And after translation:
(N-terminus) His His His His His His (C-terminus)
Since you want your His-tag sequence in the reverse primer, I assume that you want to have your His-tag at the C-terminus of your protein.
So now, look again at the double-stranded DNA. For the reverse primer we are interested in the lower sequence from 3' to 5' (complement).
3'-GTA GTA GTA GTA GTA GTA-5'
Now, since the companies that synthesize the primers for you usually demand the sequence from 5' to 3' you also need to reverse this complement sequence (reverse complement):
5'-ATG ATG ATG ATG ATG ATG-3'
So, your sequence seems to be correct and you also have a stop codon at the end of the His-tag. Just a question, are you planing to clone with restriction enzymes? I'm just wondering about your 5'-CCCGGG...-3' overhang.
BTW, you'll find programs that do this automatically for you when you google "reverse complement" for example.
Now, I'd also suggest you to use some programs that will facilitate the primer design and the cloning. Maybe you can get your hands on some programs in your research group?
Dear Adrian Marc Hauri
Thank you for your answer.
my Revers primer :
5'- CCCGGG TTA ATG ATG ATG ATG ATG ATG AGTTAAACA -3'
Restriction site : cccggg stop codon : tta His-tag : atg sequnece : agttaaaca
I am plannig to clone with restriction enzymes. my vector (pBI121) has XmaI Restriction site. so i have to use it.
I see, but does this restriction enzyme not need an overhang? NEB will usually list appropriate overhangs for the restriction enzymes. Without overhang the restriction might not work.
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