... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide... Transform TOPO Cloning Reaction into Competent Cells: You can use your standard lab protocol for this...

... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...service to further contribute to our mission to “accelerate research and discovery.” Here are the details...

...ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...

...illumination with violet light and (2) exposure to increased intracellular calcium. It has been applied to...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version named...

...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...genes essential for West-Nile-virus-induced cell death." Cell reports 12.4 (2015): 673-683. PubMed PMID:... (and multiple) genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems ... build and constantly improve gRNA libraries, excellent resources with effective gRNAs are publicly available...perturbations and applications in functional genomics, cell reprogramming, and beyond. For further details, ...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials... protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of...disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant...which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB...broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon...Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly...

...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained... brain, robust single-cell labeling is necessary to distinguish individual cells. The Brainbow-1.0 construct... May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... both of Harvard’s Department of Molecular and Cellular Biology, harnessed these attributes of Cre-lox...

... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ... the EMX1 locus in human embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target...off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity... researchers to make precise edits in mammalian cells and may decrease worries about off target effects...

...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ...better than a nitrocellulose membrane. If you have already run your blot using nitrocellulose, you may decide...protein of interest in a complicated solution like cell lysate. But they can be a lot of work — and what...instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution...

... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the... most relevant to plasmids: Genetics & Heredity, Cell Biology, Biotechnology & Applied Microbiology, Biochemistry...

...phosphorylation." Nature cell biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., ...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...commonly cited that approximately one-third of cellular proteins are modified through phosphorylation ...translation of many stop codons and detrimental effects on cell growth. The C321.ΔA strain was genomically recoded...is particularly interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion...

... living plant cells." (2013). PubMed PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...fluorescent proteins available. Fortunately, several excellent reviews have collated data from various papers... alters the FRET efficiency. Detecting certain cellular changes with an intermolecular probe is often ...Miyawaki et al., 1997), was designed to measure intracellular calcium and published in 1997. Since that time...of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments can probe...

... the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating Gene function by... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...stain the cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue...specific cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the...techniques to alter gene expression or to label specific cells, but there are too few resources to teach students... in tissue. This is useful for labeling specific cell types and for viewing how gene expression changes...

...lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell. To make...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis... cloned into E. coli cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed... proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity.  First, cut...with live host cells Now you can proceed with incubating this membrane with your cells of interest and...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...successfully infected/expressing cells by flow cytometry. The isolated cells were injected into mice and imaged...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...

...red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first... question about what should go into a cell, hover over the cell and instructions will pop up. When you... into the appropriate full and partial sequence cells. *Note: If you believe you need to deposit a collection...

... DC. 2. President's Council of Advisors on Science and Technology [PCAST] (2012). Engage to Excel: Producing...research experiences to undergraduate students (1, 2), but educators face significant challenges in designing... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...lack uracil, but contain methionine. (This allows cells carrying nonfunctional as well as functional MET... is available at our Investigations in Molecular Cell Biology Website. Many thanks to our guest blogger...right) has taught courses in biochemistry, molecular cell biology and genetics at Boston College. She is particularly...

...neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression of molecular...specific cell type. Find some transduction characteristics of select AAV serotypes in Table 2 from the...in their cell bodies. Optical stimulation in a different brain area can then reveal the cells bodies of...with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route of administration...transduction, the viral capsid proteins bind to the target cell’s surface proteins and this interaction determines...by restricting expression to genetically defined cell types. Specific transgene expression can be also...different approaches can increase the specificity of cell targeting even further. For example, injecting AAVs...

...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...and function of cellular systems. FPs have been used to track protein localization, cell structure, intracellular...associated with cellular organelles, scientists have been able to study many cellular processes, including...to fluorescently tag markers of cellular structures in mammalian cells. These plasmids use fluorescent...Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15(1) (2016...

...pH visible at the sample interface upon thaw (Fig. 2). Before thawing, all the samples were at the control...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...measure the pH of our samples, we used a solution of cell culture medium, which is buffered and contains the...