siRNA / miRNA gene silencing Human Primary Human Hepatocytes

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

What is the optimal concentration for primers in qPCR? My total volume is 20μl per reaction.

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.

Get tips on using GeneJET RNA Purification Kit to perform AAA for reviews

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Piscirickettsia salmonis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Lactobacillus brevis

Get tips on using GeneJET Plasmid Miniprep Kit to perform Plasmid Isolation Bacillus smithii

Get tips on using GeneRead DNA FFPE Kit to perform DNA isolation / purification Tissue - FFPE samples

Get tips on using GenElute™ Plasmid Miniprep Kit to perform Plasmid Isolation S. cerevisiae

Get tips on using GenElute™ Mammalian Total RNA Miniprep Kit to perform