Hi,
Here I explain my question more. Im a molecular biologist in struggle. I have a procedure and there are 2 different vectors derived from Ecoli and and an insert which will be put into an Ecoli for recombinant protein production in the end.
I cant understand why someone would insert a gene into a bacterial plasmid, then remove and add to another bacterial plasmid when these two plasmids are both derived from Ecoli.
I dont understand what subcloning is and what would happen if i take my insert and add to an expression vector directly.
Any help appreciated.
Thank you
Hi Sener,
I guess you can insert your desire gene in a vector directly; however, somebody does sub-cloning to transfer needed tag with the insert from the first vector, sometimes. Hope that would help to clarify your concern.
Hmmm. Thank you for the answer! But in my case there isn't any added tag. This procedure I have, puts the insert into a desired vector, then removes it and adds to another vector. I dont see what I would lose if I take my insert and put it to second vector directly.
What is the benefit of subcloning?
Does anyone know other than tagging, why would someone need subcloning in the different plasmids derived from same bacteria?
It depends a lot on the specifics of each situation. There are specialized cloning vectors with niceties such as selection schemes, high miniprep yields, cheap antibiotic selection, etc. and these are sometimes used first before subcloning into an expression vector (Cloning directly into an expression vector is a bit risky if the insert may be expected to be toxic and/or repression may be leaky). Sometimes it happens serendipitously; you may have an old construction bearing an insert that now needs to be transferred into a Gateway entry vector. And so on; there isn't a single overriding cause.
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