Protein expression and purification Bacteria Bacillus subtilis

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Synechococcus elongatus

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Salmonella enterica

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Pseudomonas aeruginosa

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Vibrio cholerae

Get tips on using CelLytic™ B Cell Lysis Reagent to perform Protein isolation Bacteria - Escherichia coli

Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

Get tips on using GenElute™ Bacterial Genomic DNA Kit to perform DNA isolation / purification Bacteria - Gram positive Actinomycytes

Get tips on using GenElute™ Bacterial Genomic DNA Kit to perform DNA isolation / purification Bacteria - Gram negative E.coli

Get tips on using NucleoSpin® RNA/Protein to perform RNA isolation / purification Tissue - Rat Hippocampus