I designed primer to clone the target gene with RBS, his tag.
I want to confirm below:
1. if Is it okay that extra sequence longer than target gene part??
2. I designed as RBS located after his tag sequence
But should I put RBS first and. His tag sequence later????
Hi,
1. Sequence of the target gene part should be at least half size of the primer sequence (assuming that your DNA template does not contain the RBS and His-tag sequences). This is to increase the specificity of the primer to anneal at the correct sequence location of your template.
2. The His-tag is to be linked to your protein of interest. In your current design, the His-tag won't be expressed, since it is before the RBS. Relocate His-tag to after the RBS, and remove the start codon of your target gene.
3. Besides, it is recommended to add 3 random bases before your XbaI, and BamHI restriction site of your primers to improve the efficiency of digesting your cloned gene later.
https://www.neb.com/en-sg/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
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