Sub-cloning
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Sub-cloning is the method by which a construct containing genetic information is transferred from one platform to another. The transferred genetic information (also known as an insert) may be a gene, regulatory elements, DNA or RNA structural elements, or anything which can be encoded via the medium of choice (DNA or RNA).
There are four critical steps for subcloning.
- Extraction of the genetic information.
- Preparation of destination platform.
- Recombination
- Transformation
[edit] Example Case: Bacterial plasmid subcloning
In this example, a gene from mammalian gene library will be subcloned into a bacterial plasmid (destination platform). The bacterial plasmid is a piece of circular DNA which contains regulatory elements allowing for the bacteria to produce a gene if it is placed in the correct place in the plasmid. The production site is flanked by two restriction enzyme cutting sites "A" and "B" with incompatible sticky ends.
The mammalian DNA does not come with these restriction sites, so they are built in by overlap extension PCR. The primers are designed to put the restriction sites carefully, so that the coding of the protein is in-frame, and a minimum of extra amino acids is implanted on either side of the protein.
Both the pcr product containing the mammalian gene with the new restriction sites and the destination plasmid are subjected to restriction digestion, and the digest products are purified by gel electrophoresis.
The digest products, now containing compatible sticky ends with each other (but incompatible sticky ends with themselves) are subjected to ligation, creating a new plasmid which contains the background elements of the original plasmid with a different insert.
The plasmid is transformed into bacteria and is checked by DNA sequencing.
