What is subcloning used for?
In molecular biology, subcloning is a technique used to move a particular DNA sequence from a parent vector to a destination vector. Subcloning is not to be confused with molecular cloning, a related technique.
What is DNA subcloning?
Subcloning is a basic procedure in molecular biology for transfer of DNA inserts from one vector to another to gain functionality to study the sequence of interest. Then, you screen the transformed cells for the inserted DNA.
How do you do a subcloning?
There are four steps in the subcloning process: obtain the target fragment, connect enzyme vector and target fragment, transform in host cell, identify and screen. Choose an appropriate restriction enzyme to cleave the target fragment from the vector.
What exactly is PCR used for and why is it an effective and important technique?
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail.
What is the purpose of colony PCR?
Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct.
What is digestion of DNA?
Restriction Digestion is the process of cutting DNA molecules into smaller pieces with special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes or RE’s).
How can PCR product be cloned into a vector?
Typically, a PCR reaction is performed to amplify the sequence of interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to transformation. Early PCR cloning often used Taq DNA Polymerase to amplify the gene.
Why do we use two different restriction enzymes?
The use of 2 different enzymes makes self ligation of the vector impossible and makes the insertion unidirectional. Whereas in the case of single digest, selfligation occurs and insertion may occur in both ways.