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DNA ligation: Difference between revisions
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==Abstract== | ==Abstract== | ||
This is a [[Jamesh008:consensus protocol|consensus protocol]]. This protocol describes cloning into linearized plasmid vectors and subsequent transformation. Ligation (the joining together of two bits of DNA) involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. T4 DNA ligase is used to join the DNA fragments. This enzyme will ligate DNA fragments having blunt or overhanging, cohesive, 'sticky' ends.<br> | This is a [[Jamesh008:consensus protocol|consensus protocol]]. This protocol describes cloning into linearized plasmid vectors and subsequent transformation. Ligation (the joining together of two bits of DNA) involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. T4 DNA ligase is used to join the DNA fragments. This enzyme will ligate DNA fragments having blunt or overhanging, cohesive, 'sticky' ends. Typically, it is easier to ligate fragments with complementary sticky ends than doing a blunt-end ligation.<br> | ||
[[Wikipedia:DNA ligase|DNA ligase]] is used to create a [[Wikipedia:phosphodiester bond|phosphodiester bond]] between the 5' phosphate and 3' hydroxyl groups of DNA. Most commonly, one needs to insert a DNA sequence of interest into a plasmid, ready for [[transformation]] into [[competent cells]]. Ideally, DNA and vector are individually cut with the same restriction enzyme, then both are added to a ligation reaction to be circularised by DNA ligase. [[T4 DNA ligase]] is the most commonly used [[Wikipedia:DNA ligase|DNA ligase]] for molecular biology techniques.<br> | [[Wikipedia:DNA ligase|DNA ligase]] is used to create a [[Wikipedia:phosphodiester bond|phosphodiester bond]] between the 5' phosphate and 3' hydroxyl groups of DNA. Most commonly, one needs to insert a DNA sequence of interest into a plasmid, ready for [[transformation]] into [[competent cells]]. Ideally, DNA and vector are individually cut with the same restriction enzyme, then both are added to a ligation reaction to be circularised by DNA ligase. [[T4 DNA ligase]] is the most commonly used [[Wikipedia:DNA ligase|DNA ligase]] for molecular biology techniques.<br> | ||
The two components of the DNA in the ligation reaction should be equimolar and around 100ug/ml. If the plasmid vector to target DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If too low then the result may be an excess of linear and circular homo- and heteropolymers.<br> | The two components of the DNA in the ligation reaction should be equimolar and around 100ug/ml. If the plasmid vector to target DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If too low then the result may be an excess of linear and circular homo- and heteropolymers.<br> | ||
Most commonly, following ligation the circularised plasmid, now containing your insert DNA, is transformed into competent bacteria for further selection and analysis. In bacteria, transformation refers to a genetic change brought about by taking up and expressing DNA i.e. your plasmid construct, and competence refers to the state of being able to take up DNA; most bacteria are not naturally transformable, but are made permeable to the plasmid DNA by chemical or electrochemical means. Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.<br> | Most commonly, following ligation the circularised plasmid, now containing your insert DNA, is transformed into competent bacteria for further selection and analysis. In bacteria, transformation refers to a genetic change brought about by taking up and expressing DNA i.e. your plasmid construct, and competence refers to the state of being able to take up DNA; most bacteria are not naturally transformable, but are made permeable to the plasmid DNA by chemical or electrochemical means. Competent cells are extremely fragile and should be handled gently, specifically kept cold and not vortexed. The transformation procedure is efficient enough for most lab purposes, with efficiencies as high as 109 transformed cells per microgram of DNA, but it is important to realize that even with high efficiency cells only 1 DNA molecule in about 10,000 is successfully transformed.<br> | ||
During “transformation,” a single plasmid from the ligation mixture enters a single bacterium and, once inside, replicates and expresses the genes it encodes. One of the genes on the pCX-NNX plasmid leads to ampicillin-resistance. Thus, a transformed bacterium will grow on agar medium containing ampicillin. Untransformed cells will die before they can form a colony on the agar surface. | During “transformation,” a single plasmid from the ligation mixture enters a single bacterium and, once inside, replicates and expresses the genes it encodes. One of the genes on the pCX-NNX plasmid leads to ampicillin-resistance. Thus, a transformed bacterium will grow on agar medium containing ampicillin. Untransformed cells will die before they can form a colony on the agar surface. | ||
==Materials== | ==Materials== | ||
