CRISPR Mouse Deletion SL4

Get tips on using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® to perform RNA sequencing Mouse - Bone marrow-derived macrophages (BMDMs)

Get tips on using MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), FITC, eBioscience™ to perform Flow cytometry Anti-bodies Mouse - MHCII

Get tips on using ShRNA CD24 Lentiviral Transduction Particles (CD24-V2LHS_71909)) to perform shRNA gene silencing Mouse - Prostate cancer cell lines (DU145 and PC3) CD24 lentiviral particles

Get tips on using ShRNA CD24 Lentiviral Transduction Particles (CD24-V2LHS_71908) to perform shRNA gene silencing Mouse - Prostate cancer cell lines (DU145 and PC3) CD24 lentiviral particles

Get tips on using CD29 (Integrin beta 1) Monoclonal Antibody (eBioHMb1-1 (HMb1-1)), APC, eBioscience™ to perform Flow cytometry Anti-bodies Mouse - CD29/β1-Integrin

Get tips on using MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), eFluor 450, eBioscience™ to perform Flow cytometry Anti-bodies Mouse - MHCII

Get tips on using BLOCK-iT™ Adenoviral RNAi Expression System, pAd/BLOCK-iT™-DEST RNAi Gateway Vector to perform shRNA gene silencing Mouse - P19 Foxm1

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.

Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.

Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.