...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...copies of RPP30 in diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

...156–159. https://doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method...are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is degraded...RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and...before RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues...keeping your samples cold until homogenization and cell lysis. This can be challenging depending on your...developed ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo...solutions that work by inhibiting RNase activity within cells or tissues- they don’t actually chemically stabilize...

...Antibody Fragments. Antibodies (Basel). 2019 Apr 9;8(2):28. doi: 10.3390/antib8020028. PMID: 31544834; PMCID...in transfected eukaryotic cells, allowing for them to be used as intracellular inhibitors.  Aptamers are...the kids say) affinity reagents deserved to be celebrated, there are many other amazing affinity reagents...small enough to be expressed in bacterial and other cell systems. Since the majority of these alternative...the constant region. Nonspecific binding, caused cell receptors for the constant region, is also reduced..., peptides, amino acids, drugs, metal ions, and cells. They can be used either with or as fluorophores... may sound. Validated antibodies are still an excellent option for many applications, and frankly, if ...

...beginning Let’s take a quick flash back to 2004. “Shrek 2” was the most popular movie in theaters, the Razr ...amazing years in front of us. So in our last celebratory moment, we wanted to walk through some of our...Facebook and Gmail, were launched. And Melina Fan, a cell biology graduate student, was frustrated by the ...to receive the Massachusetts Nonprofit Network Excellence Award for our electronic MTA system. We also ...now our Sr. Director of Production) pie-fully celebrating Addgene's 10 year anniversary.   Expanding...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after ...Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace with fresh media. ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container..., 5% CO 2 for ~20 h. Prepare a mixture of the three transfection plasmids into OptiPro SFM: Reagent Amount...

...phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust...sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH...monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to...recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration ...antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type...conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ... channel pipette 20–200 µL single channel pipette 2–20 µL single channel pipette Reagents Aspirating pipette...

...localizations. Figure 2: Split-wrmScarlet labeling proteins with different subcellular locations. Image from...determine cell cycle length by examining the ratio of blue to red cells. Cells undergoing rapid cell cycles...engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other cellular protein. They used...’s difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues... BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3 show...H2B-FT consistently predicted the cell cycle speed across these different cell types. In one example, H2B-FT...

... already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step...Calcium phosphate Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic...identify and enrich positive cells or a selection marker to generate stable cell lines. Transient or stable...and enrich positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and...wide variety of mammalian cell lines. Useful for difficult to transfect cell types and can be used in ...protein are degraded within the cell.   Delivery methods for mammalian cell lines As mentioned above, the...components into your target cells. Delivery of these components into mammalian cell lines is quite broad and...

... Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563. doi: 10.3390/ijms24021563 Resources on Addgene.org...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (2013). ...patient’s own cells that have been modified ex vivo, previously edited healthy donor cells can be delivered...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (2013). Gerald Schwank et al. ...on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis ...delivery directly to the patient. Patient-derived cells can alternatively be harvested, edited ex vivo, ...

... tabular HTML formatted output. Surprisingly only 2 tools provide FASTA output. A few notable tools allow...Nature biotechnology(2014). PubMed PMID: 24880488. 2. Hsu, Patrick D., et al. "DNA targeting specificity...the research community. I’ve likened it to the celebration surrounding the piñata. From the perspective ...

...C-terminus of human codon-optimized  cryptochrome 2 (CRY2) from Arabidopsis thaliana. When exposed to ...illumination of neighboring cells evokes action potentials (APs) in patched cells expressing wild type CoChR...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here...28646204. soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego ...

...dimerization-dependent protein interactions. FEBS letters, 591 2, 415-424. PubMed PMID: 28032891. This is a different... expressed in cells via plasmids or by the creation of stable cell lines and these cells are then grown... complex. Not performed in a native cell context. For AP-MS, cells are lysed before the bait protein is...and interacting proteins in mammalian cells. The Journal of cell biology. PubMed PMID: 22412018. PubMed...against PPIs that aren't stable outside the native cell context. The Split-BioID method Enter Split-BioID...tagging of proteins in both BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein...proteins that interact with the bait in a native cell contact. Other PPI methods may require protein interactions...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...CRISPR-Cas12b for mammalian genome engineering." Cell discovery 4.1 (2018): 63. PubMed PMID: 30510770....

...still depolarize neurons at lower MOIs. Figure 2: ChRgers are delivered by intravenous injection into... channels in living cells. Multiple properties of a channelrhodopsin, such as cell localization, kinetics...

...main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying gene...Different AAV serotypes have preferences for specific cell types. For example, AAV1 and AAV9 serotypes have...most cases with VSV-G that generally mediates broad cell tropism and has a high percentage of neuronal targeting...

...method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer, TU/mL N = Number of cells transduced...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with ...,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 ...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...added to 150,000 cells resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent...

...Components." Cell (2016). PubMed PMID: 27160350. Find Plasmids from the publication at Addgene 2. Green, ...circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable for up to...possible and submitted our manuscript to the journal Cell just six weeks later. The Zika publication timeline...of Toronto. His research is focused on developing cell-free synthetic biology applications for human health...de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939. PubMed PMID: 25417166. PubMed...Keith, et al. "Paper-based synthetic gene networks." Cell 159.4 (2014): 940-954. PubMed PMID: 25417167. PubMed...

...protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this...Heat block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker...running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002 20X TBS Tween...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and prepare...in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix...the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top Stack to one...

...unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4...currently doing this using two approaches: single-cell RNA-sequencing to identify neuronal class and deep...WDDD aims to acquire quantitative data on all the cell divisions of the C. elegans embryo when all essential...Wang et al., 2018).” With the split system, only cells that have both halves of cGAL can drive expression...

...systems to defend against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the...origin of cellular life. His lab has long hypothesized that life started with RNA and a simple cell membrane... circular RNA they have identified resulted in a cell migration defect. They have created a circRNA website...