Difference between revisions of "20.109(S22):M2D4"
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==Protocols== | ==Protocols== | ||
− | + | ==Part 1: | |
+ | Our communication instructor, Dr. Prerna Bhargava, will join us today for a discussion on writing figure titles and captions. | ||
====Confirm sgRNA_target with sequencing==== | ====Confirm sgRNA_target with sequencing==== | ||
Revision as of 17:55, 24 January 2022
Contents
Introduction
Protocols
==Part 1: Our communication instructor, Dr. Prerna Bhargava, will join us today for a discussion on writing figure titles and captions.
Confirm sgRNA_target with sequencing
The sgRNA_target sequence that was inserted into the expression plasmid was confirmed using DNA sequencing. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. 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 and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides, or chain-terminating bases. 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 sequence due to the presence of fluorescent dyes, one color linked to each dideoxy-base. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected.
Reagents list
Next day: Prepare for induction of CRISPRi system