PCR

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - primary human pulmonary artery smooth muscle cells

Get tips on using VWR Life Science RiboZol™ RNA Extraction Reagent to perform RNA isolation / purification Cells - primary rat brain microvascular endothelial cells

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - HeLa ChaC1

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.

Get tips on using pwPICZalpha-DT390-bi-pIL-2-Non-N-Gly to perform Protein Expression Eukaryotic cells - P. pastoris Porcine IL-2 fusion toxins

Get tips on using Flp-In™ T-REx™ 293 Cell Line to perform Protein expression and purification Mammalian cells - CAL-51 BRCA1

Get tips on using Jump In™ T-REx™ HEK 293 Kit to perform Protein expression and purification Mammalian cells - HEK 293 HER2

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

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

Get tips on using Pierce™ BCA Protein Assay Kit to perform Protein quantification Mammalian cells - PC-12