crispr-human-repression-hpv-16-e7

Get tips on using HphI NEB#R0158 to perform Restriction Enzymes HphI

Get tips on using HpyCH4III NEB#R0618 to perform Restriction Enzymes TaaI / HpyCH4III

Get tips on using HpaII NEB#R0171 to perform Restriction Enzymes HpaII / MspI

Get tips on using HphI (10 U/µL) to perform Restriction Enzymes HphI

Get tips on using pPICZ-mEGFP-hPORCN to perform Protein Expression Eukaryotic cells - HEK293 mEGFP-hPORCN

Get tips on using TaaI (HpyCH4III) (10 U/µL) to perform Restriction Enzymes TaaI / HpyCH4III

Get tips on using HpyF3I (DdeI) (10 U/µL) to perform Restriction Enzymes DdeI / HpyF3I

Get tips on using HapII (HpaII, MspI) restriction enzyme to perform Restriction Enzymes HpaII / MspI

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.