I would like to subclone in a 25 amino acid group into various intracellular domains of a GPCR that I am studying. I will be making about 8-10 constructs and I will screen for functionality using an assay that I have.
I am new to cloning so I just wanted some advice on which cloning strategy would be the most optimal.
Site-directed insertion (http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91) is a easier and efficient way to generate these clones. You can share the coding sequence of 25 aa into two over-lapping primers that can be synthesied easily like that of normal PCR primers. The attached lab protocol might be easier for you to follow.
I wouldn't clone that. The only reason to clone it would be if you really want to learn cloning, and are looking for a project to learn it with. You should have it made by de-novo synthesis. Some vendors make it cost-effective to generate variants synthetically -- they basically synthesize the whole thing once and then swap the variants in as needed. But even if that's for some reason not an option you have to order 10 genes from scratch, that's still probably no more than a $10,000 fixed, known cost. If it starts with teaching you cloning from scratch, the time and total cost can be... pretty much unbodunded :)
I agree with John, unless you are set on learning creative cloning strategies, de-novo synthesis is a more straightforward path. Say your GPCR + insert is 1200nt, that would cost $250 to make as a gBlock Gene Fragment via IDT. Going the synthesis route also allows you to design in clever cloning sites in anticipation of future changes (e.g. updating your 25 aa insert, etc.). Depending on your time, goals and the way you design it, you may not to need order 10 distinct gBlocks - this would reduce your costs while also allowing an easier time with subcloning.
https://www.idtdna.com/pages/products/genes/gblocks-gene-fragments
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