Site Directed Mutagenesis (SDM) Mouse 3T3-L1

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion MCF-7 AHR

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation H1299 PDHA1

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion K562 c-Myb gene

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Monkey - Point mutation Cos-7 PAH

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Hamster - Point mutation BHK-21 DENV4

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation U-87MG MEF

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation SH-SY5Y FGF1

Wound healing assay can be challenging due to inconsistencies and variations while making a wound on the confluent cell monolayer, consequently leads to wounds of varying sizes and widths. Moreover, this assay causes damage to the cells that are at the edge of the wound, which can prevent cell migration into the wound site and healing. The best solution is to use the standard wound healing assay kits using either combs or inserts to make a defined wound field or gap and prevent the well-to-well variation in these assays.

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

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.