Protein expression and purification Mammalian cells HEK 293

Get tips on using HEK GM to perform Mammalian cell culture media HEK

Get tips on using Nitrocef disks to perform Reporter gene assay β-lactamase substrates - HEK 293 & HEK 293T cells

Get tips on using Freedom™ DG44 Kit to perform Protein expression and purification Mammalian cells - CHO-K1 G12

DNA damage assay is a standard method for determining in-vivo/in-vitro genotoxicity by measuring the breaks in the DNA chain of animal and plant cells. Initial DNA damage leads to cell cycle arrest and, at the final stages, leads to induction of senescence or cell death (apoptosis, necrosis, autophagy, or mitotic catastrophe). Detection of DNA damage from mild to moderate to severe is challenging when studying genotoxicity in the pool of cells. It is favorable to use DNA damage assay kits available for prominent identification of the extent of damage in the analysis.

Get tips on using Bac-to-Bac™ Baculovirus Expression System to perform Protein expression and purification Insect cells - Sf9 Drosha

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 have 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 efficacy of transduction and shRNA on its target site.

Get tips on using pMT/BiP/V5-His A, B, & C Drosophila Expression Vectors to perform Protein expression and purification Insect cells - S2 HER2

Get tips on using psiCHECK-2vector to perform Reporter gene assay luciferase - HEK 293 human embryonic kidney cells