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. Molecular biology is the study of biology at a molecular level. ... This stylistic schematic diagram shows a gene in relation to the double helix structure of DNA and to a chromosome (right). ... Traditionally in medicine, a vector is an organism that does not cause disease itself but which spreads infection by conveying pathogens from one host to another. ...

Subcloning is not to be confused with cloning, a related technique. Cloning is the process of creating an identical copy of an original. ...

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). A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. ... In molecular biology, gel extraction or gel isolation is a technique where viable DNA is eluted (extracted) from an agarose gel following agarose gel electrophoresis in order to isolate a specific band. ... Polymerase chain reaction (PCR) is a molecular biology technique for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. ...

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 (commonly CIP) is also added to prevent self-ligation of the destination vector. The digested destination vector is isolated/purified and amplified using PCR. A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. ... The word complement (with an e in the second syllable, not to be confused with a different word, compliment with an i) has a number of uses. ... In biology, sticky end and blunt end are the two possible configurations resulting from the breaking of double-stranded DNA. DNA exhibits a stabilizing interaction between complementary base pairs, providing specificity to the pairing of two strands of DNA. If two complementary strands of DNA are of equal length, then... In molecular biology, DNA ligase is a particular type of ligase (EC 6. ... A phosphatase is an enzyme that hydrolyses phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxy group. ...

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. In molecular biology, DNA ligase is a particular type of ligase (EC 6. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ...

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. Transformation is the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA or RNA). ... ‹ The template below has been proposed for deletion. ... Binary fission Binary fission is the form of asexual reproduction used by most prokaryotes to reproduce. ... Wht the hell is going on huh ? You want to die ? What a bullshit ?In molecular biology, a minipreparation or miniprep is a technique for extracting small amounts (100 ng to 5 μg) of plasmid DNA from transformed bacteria, most commonly Escherichia coli. ...

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. So, for example, the "marker gene" could be for resistance to the antibiotic ampicillin. If the bacteria that were supposed to pick up the desired plasmid had picked up the desired gene then they would also contain the "marker gene". Now the bacteria that picked up the plasmid would be able to grow in ampicillin whereas the bacteria that did not pick up the desired plasmid would still be vulnerable to destruction by the ampicillin. A marker gene is used in molecular biology to determine if a piece of DNA has been successfully inserted into the host organism. ... A genetic screen (or simply screen) is a procedure or test to identify and select individuals which possess a phenotype of interest. ... Antibiotic resistance is the ability of a microorganism to withstand the effects of an antibiotic. ... Biosynthesis is a phenomenon where chemical compounds are produced from simpler reagents. ...

External links

• Subcloning Manual by Promega (may need to select language)