It is better to get the cassette with gene of your interest form other scientists.lot of professional skill is required to make it.
I recommend a seamless cloning method like Golden-Gate-cloning or the BioBricks system to build up your cassette. There's a huge library of components available for the latter: http://partsregistry.org/Catalog
If you, for whatever reason, have to start from scratch have a look at component homologs or similar cassettes to get a feeling for distances, spacers to include et cetera.
Hi Himanshi,
There are vectors available that contain various promoters, terminators, reporter genes wherein all you need to do is clone your gene of interest into the MCS. Try Clonetech, Promega, Invitrogen, etc for what they offer and couple that to what you need. There are even kits out there that let you make fusion products with your gene of interest...it just depends on what you want.
Best of Luck
Barry
Hey Himanshi, I think its nt that difficult to make a cassette. If you are lucky u can make it within a weeks time. But it depends what exactly you wanna make. if you want to make the cassette for over expressing it then you can use any vector available in the market, and amplify you gene of interest from the cDNA prepared from the organism you are interested in and then after digestion of the insert and vector you can ligate it followed by transformation. But if you want to use the endogenouse promoter then you ca again replace the vector promoter by your endogenous promoter (it will be done in the second step). Good Luck!!
Hi Himanshi,
Well if you want for overexpression then it is very easy venture. All you need to find your appropriate vector available commercially in the market and design a cloning strategy and go fr it...
@Goutam Tanti..I have to look for the vector having promoter, terminator and an MCS..right?
Hi Himanshi, no I meant to check vector for your priority, like what do you want..if you want to over express in mammalian (or any other eukaryotic cell lines) cell lines then you need to check for mammalian expression vector like pCDNA etc, and then what you need is whether you want to study localization or not then you look for the tag present in the vector (GFP, myc, FLAG)...etc all the expression vector generally have the promoter, MCS poly A signal (equivalent to terminator) etc. good luck!!
I'd recommend fusion PCR, that should be doable with minimal links in between respective parts
If you have all the components in plasmds. fusion PCR and/or Gibson cloning is the way to go. If you do not have the components, easiest is to get it synthesized by outsourcing to a company.
Check out https://www.sigmaaldrich.com/technical-documents/articles/biology/aavs1-safe-harbor-landing-pad-cell-lines.html
Hope this helps!
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