Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.
Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.
DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
Contamination can affect cell characteristics, i.e., growth, metabolism, and morphology leading to unreliable and erroneous experimental data. Depending on the source of contaminants, one can detect contamination by using a light microscope, gram stain, isothermal amplification, or PCR. Bacteria and fungi can usually be identified by optical microscopy. Mycoplasma in cell cultures cannot be detected visually. Hence, these microbes can go unnoticed for long periods and are determined using dedicated assays. Early and rapid identification of contaminants is vital to detect, handle and prevent contamination for good cell-culture practices. However, detection and identification can be challenging and tricky based on usual visual identifications. Hence it is essential to use a standard contamination detection kit to detect and maintain best practices.
DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.
DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.
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