Protein expression and purification Bacteria Escherichia coli

Get tips on using pS4E8Y to perform Protein Expression Prokaryotic cells - E. coli SELP

Get tips on using pS2E8Y to perform Protein Expression Prokaryotic cells - E. coli SELP

Get tips on using pEThCRM to perform Protein Expression Prokaryotic cells - E. coli CRM197

Get tips on using pCRM197 to perform Protein Expression Prokaryotic cells - E. coli CRM197

Get tips on using pIG6 to perform Protein Expression Prokaryotic cells - E. coli Ppolcp19k

Get tips on using pRSF1b to perform Protein Expression Prokaryotic cells - E. coli AfFtn

Get tips on using pIBAINS to perform Protein Expression Prokaryotic cells - E. coli hInsulin

Get tips on using pTHAO1 to perform Protein Expression Prokaryotic cells - E. coli hAOX1

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 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.