Protein expression and purification Mammalian cells HEK 293

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DNA isolation and purification is the first critical step in sample preparation that helps ensure optimal performance of downstream assays like PCR, microarrays, and sequencing. Failure in yielding high-quality DNA would be the major reason that DNA doesn't work for the downstream application. To circumvent this, one should follow the recommended storage conditions to minimize DNA degradation by nucleases and shouldn't overload the purification system.

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

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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 T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Renal tissue

Get tips on using T-PER™ Tissue Protein Extraction Reagent to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - Mouse macrophages

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - Neuro 2a

Get tips on using Pierce™ Coomassie (Bradford) Protein Assay Kit to perform Protein quantification Mammalian cells - OUMS-27