CRISPR Mouse Deletion Neuro 2a

Protein ladders are a set of standards known as molecular weight proteins that are utilized to identify the approximate size of a protein molecule run on a PAGE gel electrophoresis. The challenges in running the ladders are the choice of appropriate protein standard as it is used as visual evidence of protein migration, transfer efficiency, and positive control. Suitable protein markers can be selected on the basis of required properties and applications, i.e., fluorescent ladder, IEF, 2D SDS-PAGE ladder, natural ladder with an isoelectric point, and optimized ladders for Western Blot chemiluminescence detection. The key factors for running a distinct protein ladder are buffer conditions, charge/voltage at migration time, and the gel's concentration.

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. Multiplexing such a reaction amplifies the design challenges where one target requires 3 primers, which should be exclusively bound nowhere in the template DNA or to each other. Similarly, two targets require 6, three require 9, and so on. Each amplicon needs to be either a different size (for gels) or labeled with a different fluorescent tag that is spectrally distinct from the others in the reaction. Further complicating this, different targets in the reaction can compete with each other for resources and causes more challenges in the detection of amplicons. However, with proper primer designing, their validation, optimize quality and concentration of the enzyme and buffers certainly lead to a successful multiplex PCR reaction.

A PCR reaction consists of the template DNA, two primers covering the amplification site, an enzyme, and buffers. Multiplexing such a reaction amplifies the design challenges where one target requires 3 primers, which should be exclusively bound nowhere in the template DNA or to each other. Similarly, two targets require 6, three require 9, and so on. Each amplicon needs to be either a different size (for gels) or labeled with a different fluorescent tag that is spectrally distinct from the others in the reaction. Further complicating this, different targets in the reaction can compete with each other for resources and causes more challenges in the detection of amplicons. However, with proper primer designing, their validation, optimize quality and concentration of the enzyme and buffers certainly lead to a successful multiplex PCR reaction.

RNA quantification for appropriate concentration and quality (260/280 ratio) is an important step before downstream analysis (including sequencing, RT-qPCR, etc.). Having insufficient RNA quantities or a high salt or phenol in the RNA product can lead to variable or irreproducible downstream results. The various methods used for RNA quantification include: 1. UV spectrophotometric (challenges include: low sensitivity, cannot distinguish between nucleic acid species), 2. Fluorescence-based (challenges include: requires standards, cannot measure amplifiability, not sequence-specific), and 3. RT-PCR (challenges include: requires standards, time-intensive, costly). In order to overcome these challenges, and also to ensure the proper quantification and quality control for RNA product, it is important to use at least two or more methods in order to discard any inconsistencies. Using standards for calibrations increases the sensitivity range for RNA detention (fluorescence- and RT-PCR-based methods). When using RT- PCR, it is important to choose correct primers, aligning to the desired site on the template and of appropriate product length, along with positive, negative and loading controls. It is also important to have at least two primer pairs in order to confirm results.

RNA quantification for appropriate concentration and quality (260/280 ratio) is an important step before downstream analysis (including sequencing, RT-qPCR, etc.). Having insufficient RNA quantities or a high salt or phenol in the RNA product can lead to variable or irreproducible downstream results. The various methods used for RNA quantification include: 1. UV spectrophotometric (challenges include: low sensitivity, cannot distinguish between nucleic acid species), 2. Fluorescence-based (challenges include: requires standards, cannot measure amplifiability, not sequence-specific), and 3. RT-PCR (challenges include: requires standards, time-intensive, costly). In order to overcome these challenges, and also to ensure the proper quantification and quality control for RNA product, it is important to use at least two or more methods in order to discard any inconsistencies. Using standards for calibrations increases the sensitivity range for RNA detention (fluorescence- and RT-PCR-based methods). When using RT- PCR, it is important to choose correct primers, aligning to the desired site on the template and of appropriate product length, along with positive, negative and loading controls. It is also important to have at least two primer pairs in order to confirm results.

RNA quantification for appropriate concentration and quality (260/280 ratio) is an important step before downstream analysis (including sequencing, RT-qPCR, etc.). Having insufficient RNA quantities or a high salt or phenol in the RNA product can lead to variable or irreproducible downstream results. The various methods used for RNA quantification include: 1. UV spectrophotometric (challenges include: low sensitivity, cannot distinguish between nucleic acid species), 2. Fluorescence-based (challenges include: requires standards, cannot measure amplifiability, not sequence-specific), and 3. RT-PCR (challenges include: requires standards, time-intensive, costly). In order to overcome these challenges, and also to ensure the proper quantification and quality control for RNA product, it is important to use at least two or more methods in order to discard any inconsistencies. Using standards for calibrations increases the sensitivity range for RNA detention (fluorescence- and RT-PCR-based methods). When using RT- PCR, it is important to choose correct primers, aligning to the desired site on the template and of appropriate product length, along with positive, negative and loading controls. It is also important to have at least two primer pairs in order to confirm results.