Get tips on using RNeasy Protect Cell Mini Kit (50) to perform RNA isolation / purification Cells - primary human mononuclear cells
The process of RNA extraction from bacteria, in general, involves an RNA-protective, effective lysis of bacterial cell wall (which may pose difficulties). EDTA promotes loss of outer membrane to provide lysozyme with access to peptidoglycan. Another common method for cell wall lysis is mechanical disruption using a homogenizer (applied for gram-positive bacteria and some strains of gram-negative bacteria). Following lysis, it is necessary to disrupt protein-nucleic acid interactions, which can be achieved by adding sodium dodecyl sulfate (SDS). Next step involves using phenol-chloroform-isoamyl alcohol extraction, where RNA can be obtained from the bottom organic phase, the top phase consists of DNA and the interphase contains proteins. Isoamyl alcohol is an inert and optional addition to this mixture and is added as an anti-foaming reagent to reduce the interphase. Following RNA extraction, the samples should be checked for its quality by gel electrophoresis (23S and 16S rRNAs and 5s rRNA and tRNA bands) or UV spectrophotometric or fluorescence methods.
Get tips on using Gentra Puregene Cell Kit Plus (6.7 x 109) to perform DNA isolation / purification Cells - Immortalized cell lines H1 hESc
Get tips on using EGMTM-2 Endothelial Cell Growth Medium-2 BulletKit to perform Stem cell culture media hLung Mesenchymal Stem Cells (MSCs)
Get tips on using FitAmp Blood and Cultured Cell DNA Extraction Kit to perform DNA isolation / purification Cells - Immortalized cell lines SH-SY5Y
Get tips on using EBMTM-2 Endothelial Cell Growth Basal Medium-2 to perform Stem cell culture media Cord blood-derived endothelial cells(hCBiPS2)
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.
Get tips on using EasySep™ Direct Human Naïve B Cell Isolation Kit to perform Cell Isolation Naive B cell
Get tips on using MACSxpress Whole Blood Pan T Cell Isolation Kit, human to perform Cell Isolation Naive Pan T cell
Get tips on using EasySep™ Human Naïve Pan T Cell Isolation Kit to perform Cell Isolation Naive Pan T cell
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