shRNA gene silencing Mouse Prostate cancer cell lines (DU145 and PC3) CD24

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 LIVE/DEAD Fixable Violet Dead Cell Stain Kit to perform Live / Dead assay mammalian cells - mouse splenocytes

Get tips on using illustra tissue and cells genomicPrep Mini Spin Kit to perform DNA isolation / purification Tissue - kidney

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

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.

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.

Get tips on using DIA-310: Anti-CD31 (Ms) from Rat (Clone: SZ31) for mouse FFPE tissue to perform Immunohistochemistry CD31 - Rabbit Rat -NA-

Get tips on using MethylFlash™ Methylated DNA Quantification Kit to perform DNA methylation profiling Whole genome profiling - MCF-7, MDA-MB-453 human breast cancer

Get tips on using Imprint® Methylated DNA Quantification Kit to perform DNA methylation profiling Gene specific profiling - Mouse muscle stem cells SPRY1

Bacterial culture is a process of letting bacteria multiply in a controlled fashion (temperature, humidity, oxygen content or shaking), in a predetermined culture medium (antibiotic resistance to obtain homogenous clones). It is an important step, especially during cloning, as a single cell can be grown homogeneously (on semi-solid or in liquid conditions) to obtain colonies. As mentioned, bacteria can be cultured in broth cultures (Luria broth or LB) or Petri dishes (Agar plates). A specific antibiotic can be added to the broth or agar plates in order to grow bacteria which have the gene insert conferring its resistance to that antibiotic. Following points are necessary to consider for optimal growth conditions: 1. In general, most bacteria grow well at 37C, but there are some strains which require growth temperatures between 25-30C. 2. It is ideal in broth cultures to fill the flask to ⅓ or less of the total flask volume for optimal aerobic growth. 3. Shaking speeds between 140-180 rpm are appropriate to ensure aeration and that the cells are surrounded by fresh media, and do not settle.