Hi, I need some advice on primer design for Gateway cloning.
I am redesigning a forward primer in order to insert a strong Kozak sequence.
My primer is as follows:
GGGGACAAGTTTGTACAAAAAAGCAGGCTctgccgccaccatgg(+17bp)
where upper case is the GGGG + attB1 site.
Lower case is an edited Kozak incl. start side, followed by 17 gene specific bp.
My question is whether I’ve designed the primer correctly. There is no N-terminal tag to be inserted. The 17 gene-specific bp aneal directly to the area after the start site in the gene.
We are assuming the 5’ UTR is uneeded.
Any advice from the experienced cloners?
note: I already have my reverse primer, and the melting temperatures are alright for this set.
Hello,
If you are planning to express a native protein not a tagged fusion protein, the sequence seems ok.
If you need a tag on the protein, watch the AAA-AAA in the attB1 site. The AAA-AAA codes Lys-Lys so you add your tagged target sequence just after the attB1 site. Between attB1 site and target sequence, 2-mer DNA (AA,GA,AG) should be added in order to adjust the reading flame to be consecutive with Lys-Lys.
Does your 17nt gene specific sequence is directly connecting to your 2nd flame nts? If so, I think it would be ok, as far as you don't need to tag the protein.
hope it helps
Hi,
looks good. For this long primer, I would recommend inspecting the secondary structure by using program mfold. In case you observe 3'end of the primer making strong secondary structure, silent mutations may be used (but do not target silent mutations to attB).
Best, Vesa
Hi, thanks for all the input. I designed a forward primer with the edited Kozak and ended up with also designing a reverse primer with a FLAG-tag (C-terminal). Looking forward to trying it out. :) The primers ended up being about 90nt long - but from what I read this shouldn't neccessarily cause any issues.
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