2,366
edits
DNA ligation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
m
→Abstract
m (→Abstract) |
|||
| Line 10: | Line 10: | ||
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 10<sup>9</sup> 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. | 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 10<sup>9</sup> 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. | ||
During | 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== | ||
