Subcloning
From Freepedia
In molecular biology, subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality.
Subcloning is not to be confused with cloning, a related technique.
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Procedure
Restriction enzymes are used to excise the gene of interest (the insert) from the parent. The insert is purified in order to isolate it from background junk. A common purification method is gel isolation. The number of copies of the gene is then amplified using Polymerase Chain Reaction (PCR).
Simultaneously, the same restriction enzymes are used to digest (cut) the destination. The idea behind using the same restriction enzymes is to create complementary sticky ends, which will facilitate ligation later on. A phosphatase is also added to prevent self-ligation of the destination vector. The digested destination vector is isolated/purified and amplified using PCR.
Once sufficient copies are produced, the two PCR products are mixed together with DNA ligase. A typical ratio of insert genes to destination vectors is 2:1; by increasing the insert concentration, self-ligation is further decreased. After letting the reaction mixture sit for a set amount of time at a specific temperature (dependent upon the size of the strands being ligated; for more information see DNA ligase), the insert should become successfully incorporated into the parent plasmid.
Amplification of product plasmid
The plasmid is often transformed into a bacteria like E. coli. Ideally when the bacteria divides the plasmid should also be replicated. In the best case scenario, each bacteria cell should have several copies of the plasmid. After a good number of bacterial colonies have grown, they can be miniprepped to harvest the plasmid DNA.
Screening
In order to ensure growth of only transformed bacteria (which carry the desired plasmids to be harvested), a marker gene is used in the destination vector for screening. Typical marker genes are for antibiotic resistance or nutrient biosynthesis.
External links
- Subcloning Manual by Promega (may need to select language)
