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.
I have tried to fabricate Liver organoids and would like to study the impact of FBS on healthy and tumor organoids. Since the compositions of FBS is unknown, do you recommend any alternatives like Human platelet lysate, etc?
The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.
The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.
The formation of DNA from an RNA template using reverse transcription leads to the formation of double-stranded complementary DNA or cDNA. The challenges with this process include 1. Maintaining the integrity of RNA, 2. Hairpin loops or other secondary structures formed by single-stranded RNA can also affect cDNA synthesis, and 3. DNA-RNA hybrids, which may result when the first strand of cDNA is formed. For the first challenge, using workflows that involve proper isolation and storage of RNA, and maintaining a nuclease-free environment helps obtain RNA with ideal 260/230 ratios. Using a reverse transcriptase that can tolerate high temperatures (50-55oC), overcomes obstacles imposed by secondary RNA structures. Finally, RNase H has the ability to hydrolyze RNA before the formation of a second cDNA strand. It is important to ensure that RNase H activity is optimal because higher RNase H activity leads to premature degradation of the RNA template. Many reverse transcriptases offer built-in RNase H activity.
Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.
Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.
Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.
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