...occurs when bacterial cells regulate gene expression in response to changes in cell density. At the mixer...

...particles that can transduce cells. Infectious titers are typically quantified by cell transduction assays. Wild-type...

... sure that the world will go on without my whole-cell extract from 2009. Join a newsletter to stay up-...

... that your reference genes are suitable for your cell lines and/or tissue types.  Please do note my oh-so-subtle...reference genes to save on plate space. For many cell lines and tissue types, appropriate reference genes...at determining the best options for a particular cell or tissue type (such as here, here, or here).  If...your data  Once you’ve done the work to produce excellent and reliable qPCR data, you’ll of course want ...

...spatio-temporal control of gene expression within a cell. Thanks to the work of Huimin Zhao and colleagues...on a version that she hopes will change the intracellular localization of RNA. “You can think of changing...

...is immediately ready to transform into competent cells. The master mix of enzymes can be purchased from...mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically ...

...are often used to study vertebrate embryology and cell development. One major resource for frog research...Organism to Study and Model Human Genetic Disease. Cells Tissues Organs, 205(5–6), 303–313. https://doi.org...

...introduction of nucleic acids into bacteria or eukaryotic cells is a common technique in molecular biology and scientific...

...efficient gene modification. Maeder ML et al. Mol Cell. . 2008. Jul 25. 31(2):294-301. PubMed PMID 18657511...

... mechanical method before your enzyme lysis. For cell cultures, enzyme lysis is typically sufficient.  ...proteins easily available in the cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, ...while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the proprietary...expensive Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased...When choosing your lysis buffer, consider the subcellular location of your protein. Proteins located in...Membranes The two membrane options are PVDF and nitrocellulose. PVDF is a sturdy membrane that can be stripped...membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You can visualize proteins...

...Bragdon MD, Lopez Rivera F, Biette KM, DePace AH. Mol Cell. 2015 Jun 4;58(5):718-21. doi: 10.1016/j.molcel.2015.04.025...

...applications like recombinant protein purification and cell-free circuit design in E. coli - Freemont Lab MoClo-YTK...

...and the resulting heteroduplex is resolved by the cell’s mismatch repair (MMR) system. Alternatively, if...

...GCaMP6f-P2A-nls-dTomato Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox...

... chemically induce neuronal activity in specific cell types. See our Chemogenetics Plasmids and Chemogenetics...

...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...

...tweeted by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific...

...limit of detection for arg1 expressing ECN is 109 cells/ml, which is within the range of  the ~1011 commensal...

...for serotype-specific differences in tissue and/or cell type tropism. Introducing changes and/or short peptides...

...SARS-CoV-2 proteins in bacteria, yeast, and mammalian cells. We teamed up with Ginkgo Bioworks to quickly bring...

...anti-tumour therapy using antibodies targeting dendritic cells. These genetic tools were pivotal in carrying out...

...recently shared our protocol for making competent cells to save time and money. My other stingy science ...

...insertion. The linear PCR product is introduced into the cell by direct transformation. A given insert will typically...

...expressed in your target cells. For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard... CRISPR machinery. For more difficult cells (e.g. primary cells), viral delivery of CRISPR reagents may...identify and enrich positive cells, or selection marker to generate stable cell lines (for details, see Plasmids...wide variety of mammalian cell lines Useful for difficult to transfect cell types, can also be used in...possible genotypes within the resulting cell population. Some cells may be wild type due to either (1) a ...efficient target cleavage in cells expressing both Cas9 and gRNA. Edited cells may be homozygous or heterozygous...will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR ...

...200x the number of cells as plasmids in the TKO library. Using large numbers of cells minimizes the effects...selection Most cells die or do not pass selection (in the case of a reporter) Sort “winning” cells Sequence ...library Apply selection; most cells will survive Perform NGS on the surviving cells Compare NGS results between...identify those cells that do not survive the selection mechanism. You’ll infect two sets of cells and subject...libraries, plasmid DNA can be delivered directly to the cells of interest. With others, notably pooled lentiviral...subsequently used to deliver the plasmids to the cells of interest. In either case, next-generation sequencing...Pooled Library Screens In pooled library screens, cells are infected at a very low multiplicity of infection...

... produced. Cells - Antibodies can also be produced in cell cultures using hybridomas, cell cultures created...created by fusing B cells with myeloma cells (immortalized B cells). The genetic reorganization that creates...identify and sort cells from a mixed population. Overview of application Collect cells from cell culture or ...specificity occurs in the B cell itself prior to antibody production; therefore, each B cell only produces one ... specific hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express...determines the isotype of the antibody. B cells, the immune cell responsible for producing antibodies, can...mature B cell can clone itself to enable higher production of a single antibody, but each B cell can only...

...AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid 911E4 cells Cell line compatible...then transformed into AdEasier-1 cells , which are BJ5183 E. coli cells containing pAdEasy™. pAdEasy™ is... 293/911 cells throughout virus production. During experiments, GFP can be used to sort cells infected...must be transfected into 911E4 cells for virus production, as 293 cells do not contain E4. Adenovirus ...the Coxsackie-Adenovirus Receptor (CAR) to enter cells. Recombinant adenovirus has two genes deleted: E1...linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct, and...about AAV, read our AAV guide . Can I make a stable cell line with adenovirus? No, adenoviral vectors can...

...packaging cell line. γ-retroviruses can transduce target cells, but the transduced target cell does not...using helper-free packaging cell lines This method utilizes a packaging cell line that stably expresses... packaging cell line. For example, Phoenix, a second generation γ-retrovirus packaging cell line developed...and rat cells), or an amphotropic envelope, Phoenix-AMPHO (for the infection of mammalian cells.) Using...transduce both dividing and non-dividing cells because once in the host cell cytoplasm, the viral componentes ...γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted the development of multiple...Moloney Murine Leukemia Virus) or MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol...

...can use FACS to sort cells expressing GFP and later expand these cells into a cell line. Where does lentivirus...actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This... necessary. What cell line should be used in order to produce lentivirus? 293T cells are usually used ...be used to make stable cell lines? Lentiviruses can be used to make stable cell lines in the same manner...infected host cells. When these antibiotics are added to the growth medium of the host cells, they kill ...kill off any cells that have not incorporated the lentiviral genome and those cells that survive can be expanded...plasmids are generalized and appropriate for varied cell types and systems. When planning your experiment...

...concepts and challenges. Molecular Biology of the Cell , 30 (11), 1249–1271. PMID: 31084567 (Link opens...

... tools that effectively transduce many mammalian cell types. AAV is used in a wide range of experiments...

...with Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell Stem Cell . 17 (2), 221–232. PMID: ...in the same cell. This is called multiplexing , and increases the likelihood that any cell containing ...can increase efficiency. Synchronizing cells or modulating the cell cycle can help as HDR takes place during...expressing stable cell line. After applying your screening conditions to your transduced cell population, you...tracking of RNA in live cells, including orthogonal RNA/DNA labeling in the same cell. Figure 11: Overview...promiscuity is unique to bacterial cells and does not occur in mammalian cells. SHERLOCK , developed by the ...genomic loci in living human cells by an optimized CRISPR/CAS system. Cell . 155 (7), 1479–1491. PMID: ...

...persist in non-dividing cells for a period of years without damaging the host cell. In contrast, viral particles...through cell lysis. Episomal stability enables long-term transgene expression in non-dividing cells and is... the helper plasmid are transfected into HEK293 cells, which contain the adenovirus gene E1+, to produce...serotypes differ in their tropism, or the types of cells they infect, making AAV a very useful system for...for preferentially transducing specific cell types. The chart below gives a summary of the tropism of AAV...AAV5, AAV6, AAV9 Pancreas AAV6, AAV8 Photoreceptor Cells AAV2, AAV5, AAV8 RPE (Retinal Pigment Epithelium...displays very high infectivity across a broad range of cell types. The mutant AAV-DJ8 displays the properties...

...antigens in living cells or tissues. sdAbs can also be used to manipulate cellular functions, like in ...sdAbs over conventional antibodies include:  Excellent tissue penetration, thanks to their small size...

...al., 2022). B, Representative images of HEK293T cells and neurons expressing nLightG or nLightR before...

... repository of CRISPR screens Cellosaurus: A knowledge resource for cell lines IBEX Knowledge Base: A ...

...experiments to test nickase preferences using HEK293 cells. They started by comparing D10A and H840A total ...

...detection of dopamine in flies, fish, and mice." Cell 174.2 (2018): 481-496.PubMed PMID: 30007419. PubMed...the brain has been in vivo microdialysis of extracellular fluid (Darvesh et al., 2011). This technique... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...

...a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters. mTFP1 is ...

... expression after AAV injection, what region and cell class ended up expressing your virus? If you were...