Protein Expression Prokaryotic cells A. tumefaciens

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - HOG

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - STTG1

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - Rat_Circumvallate papillae

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - Caco-2

Get tips on using Subcellular Protein Fractionation Kit for Cultured Cells to perform Protein isolation Mammalian cells - SH-SY5Y

Get tips on using pPICZα A, B, & C Pichia Vectors to perform Protein expression and purification Yeast - Pichia pastoris EDIII-D1

Get tips on using PureYield™ Plasmid Miniprep System Technical Bulletin to perform Plasmid Isolation Agrobacterium tumefaciens

Get tips on using FreeStyle™ 293-F Cells to perform Protein expression and purification Mammalian cells - HEK 293 EGFR

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.

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.