Molecular Cloning Central
The key principles of molecular cloning were discovered a little over 50 years ago. Since then, molecular cloning has become one of the most powerful tools of the molecular biology laboratory enabling the expression of the smallest genes, as well as the engineering of whole genomes.
Welcome to cloning central - a centralized portal for molecular cloning techniques, protocols, and troubleshooting guides. Click on the links below to get started.
PCR Cloning Protocol
In a traditional protocol, reaction components are assembled as described below. The final volume should be 50 µL.
Thaw all reagents on ice
Assemble reaction mix into 50 µL volume in a thin walled 0.2 mL PCR tubes.
Add reagents in following order: water, buffer, dNTPs, MgCl2, template primers, Taq polymerase.
Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
Prepare negative control reaction without template DNA.
Prepare positive control reaction with template of known size and appropriate primers
Traditional Molecular Cloning Steps and Timeline
At best, traditional cloning may take only 2 weeks, but troubleshooting after each phase
may extend the cloning process to multiple weeks or months.
Molecular Cloning Strategies
Since the seminal discoveries of the basic principles underlying molecular cloning, a number of cloning strategies have been developed to improve on the ease and speed at which DNA fragments can be recombined. Traditional cloning, also called PCR cloning, requires the use of the polymerase chain reaction (PCR) to amplify the template sequence of interest (usually the gene of interest) and add restriction sites to the ends of the sequence; TA cloning is one of the simplest forms of cloning. In this method, vectors containing 5' thymine overhangs are used to accept PCR products in which additional 3' adenosine overhangs have been added on by the nature of TAQ polymerase ampliﬁcation; Seamless cloning technologies eliminate the requirement for restriction enzymes. This can be advantageous when an insert contains a number of restriction sites within its sequence, making it difficult to identify restriction enzymes that will not cut the gene of interest during the cloning procedure.
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