Protein Expression Eukaryotic cells N. benthamiana

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 PhosphoSerine Antibody Q5 (100 µg) to perform Protein tag Detection of proteins containing phosphorylated serine residues

Get tips on using PhosphoThreonine Antibody Q7 (100 µg) to perform Protein tag Detection of proteins containing phosphorylated threonine residues

Get tips on using Cell Lysis Buffer (10X) to perform Protein isolation Tissue - Mouse lung tissue

Get tips on using 2x Laemmli Sample Buffer to perform Protein isolation Bacteria - Vibrio alginolyticus

Get tips on using TRI Reagent™ Solution to perform Protein isolation Bacteria - Vibrio cholerae

Get tips on using QuantiPro™ BCA Assay Kit to perform Protein quantification Colorimetric method

The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.

Get tips on using 300 prep FavorPrep™ Plasmid DNA Extraction Mini Kit (sample size: 1~ 5 ml culture cells) to perform Plasmid Isolation Vibrio parahaemolyticus