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| ==Introduction== | | ==Introduction== |
− | [[Image:Be109tracedata.jpg|right|thumb|160px|Sequence trace data]]
| + | The CRISPRi system involves three genetic elements: the target gene within the host genome, the pdCas9 plasmid, and the pgRNA_target plasmid. Though the target genome is native to the host cell, the plasmids must be transformed into the cell and maintained using antibiotic selection. Throughout this module, we have learned about and worked with the CRISPRi plasmids as individual units, but now we will consider the system as a whole in the context of gene regulation. |
− | [[Image:Be109dideoxynucleotide.jpg|thumb|left|450px|Normal bases versus chain-terminating bases]]
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− | [[Image:Be109sequencinggel.jpg|center|thumb|80px|Sequencing gel]]
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− | During this time, you will evaluate the DNA from your two X#Z candidates. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (see schematic above left). These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA s sequence. In the
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