Get tips on using MISSION® pLKO.1-puro Non-Mammalian shRNA Control Transduction Particles to perform shRNA gene silencing Human - Islets of langerhans Negative control (scrambled) lentiviral particles
Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.
RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.
Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.
Get tips on using GeneArt™ Site-Directed Mutagenesis System to perform Site Directed Mutagenesis (SDM) Human - Point mutation LNCaP RasGRP2
Get tips on using GeneArt™ Site-Directed Mutagenesis PLUS System to perform Site Directed Mutagenesis (SDM) Mouse - L929 T1L σ1
Get tips on using GeneArt™ Site-Directed Mutagenesis PLUS System to perform Site Directed Mutagenesis (SDM) Human - Insertion SKOV3 miR-134
Get tips on using GeneArt™ Site-Directed Mutagenesis PLUS System to perform Site Directed Mutagenesis (SDM) Human - Point mutation A498 PIK3CB
Get tips on using GeneChip® HT 3' IVT PLUS Reagent Kit to perform RNA amplification & labeling Mammalian - RNA, Human Endometrial Stromal cells Biotin
The most widely used method for protein quantification is by spectrophotometry. The concentration of the protein in the samples is measured at an absorbance of 280 nm. The absorbance of the sample protein is then plotted against a standard curve. This method allows for total protein quantification in a sample (cell and tissue extracts). Before analysing the concentration of protein in the sample, it is important to choose the right test method. For high protein concentration samples (above 5 - 160 mg/ml) the best method is to use the Biuret test. For low concentrations samples (between 1 - 2000µg/ml) the best methods are Lowry assay, BCA assay, Bradford assay and coomassie blue (for exact sensitivity of the test kits you use, refer to manufacturer's protocol). If the samples contain detergents like Triton X-100 then BCA assay is the best choice. For samples that have proteins larger than 3 KDa in size Bradford assay is the best choice. Each method has advantages and disadvantages, plan your analysis considering your sample characteristics.
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