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Reagents for Darwin Toolbox

The manipulation of DNA to create new genes or genetic elements relies on two key processes. Firstly, the amplification of DNA by a process known as the polymerase chain reaction (PCR), and secondly by the removal and reorganisation of DNA segments, digestion and ligation. These two processes make up the process known as DNA cloning.

An explanation of the principles behind PCR can be found [http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml here] or, alternatively, [http://www.youtube.com/watch?v=x5yPkxCLads]. The key enzyme in PCR is polymerase, an enzyme which joins individual nucleotides together into long strands of DNA. There are a wide variety of polymerases that exist, all with slightly different attributes, but Taq polymerase has been a reliable workhorse for research labs for many years, and the Quickload® Taq 2X MasterMix solution provides the a simple and cost-effective tool to begin your PCR reactions.

To cut out segments of DNA, a restriction endonuclease is needed. A restriction endonuclease reads a DNA strand and cuts at specific points, allowing you to reliably excise the region that you need. A detailed description of this process can be found [http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/14%20restriction%20enzymes.pdf here]. The enzymes EcoRI, XbaI, SpeI,,and PstI are a set of restriction enzymes used for BioBrick cloning. BioBrick cloning is a standard of DNA cloning used within the synthetic biology community as it can be used to reliably build novel genetic circuits with relative ease. Further information on BioBricks can be found [http://parts.igem.org/Help:An_Introduction_to_BioBricks here]. Once DNA fragments have been cut, they can be joined to one another using a DNA ligase enzyme. A ligase enzyme joins two fragments of DNA together by forming a phosphodiester bond between them, fusing them together so that they cannot break away. The Quick LigationTM Kit joins two strands of DNA together in under 5 minutes, and is in an easy-to-use format that makes it perfect for use with Darwin Toolbox.

During DNA cloning, it is often necessary to check that the fragments of DNA amplified or cut out are the correct size and to purify them away from contaminants. In order to establish their size, a DNA ladder is necessary. The 1kb DNA Ladder is a suitable reference for a wide range of DNA fragment sizes, and so is an excellent match for use in Darwin Toolbox.