...copies per cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how...Growth conditions The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum Freshly...colony and do not subculture directly from glycerol stocks, agar stabs, or liquid cultures. Freshly transformed...pairs can be separated more readily at lower temperatures and allows the replication machinery room to...if you need two plasmids for an experiment, make sure they have "compatible" ori's. See the table below... affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...

...able to eliminate the need for axenic (pure) laboratory cultures to study these microbes. From 16S rRNA...GPS coordinates, water temperature readings using a heat gun, and pH measurements. We collected small water...samples could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery... bank and the vehicle battery/alternator to make sure the vehicle battery didn't run down. In September...processing environmental samples Since the high temperatures and unstable ground around these hot springs...-term stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot...able to safely get back to our lab work. Figure 3: When a bison wanders into the campsite, leave...

...There is no known safe level of exposure to asbestos – i.e., any exposure to asbestos is potentially capable...the exposure, the greater the risk of disease) there is no known safe threshold or “safe” exposure level... goal of the Cancer Moonshot Initiative being to cure cancer, more funding and research opportunities ...unfortunately, completely preventable. Mainly caused by exposure to asbestos, mesothelioma is a form of cancer ... not quick. It can take decades from the first exposure to asbestos to the time when someone starts seeing...average latency period (time from first asbestos exposure to the time of diagnosis) is about 35 years, but... has noted, there is no safe level of asbestos exposure. Anyone exposed is at risk of developing mesothelioma...

... different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria...resistance is defined as microbial survival despite exposure to antibiotics designed to kill them or to impede...multi-drug resistance: definitions vary in the literature, but generally organisms with multi-drug resistance...contributing to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example...populations to antibiotics at unnecessary times, putting pressure on resistant bacteria to over-take the population... and making them more difficult to treat in the future. Patients not finishing the whole course of antibiotics...killed by a higher concentration, or sometimes more exposure time is needed for the drug to work… if the instructions...

...developing transgenic hairy root lines in tissue cultures takes half a year, and generating a transgenic...in the medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance...production either in the plant or in plant tissue cultures.   C. roseus is not a model organism such as Arabidopsis...used to develop stable hairy root lines in tissue culture. This method is commonly used in C. roseus, but...not reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables...overexpress a gene and evaluate its function. In nature Agrobacterium contains a plasmid with a T-DNA (... does not work well, as the leaves have a waxy texture and are difficult to infiltrate. We have recently...

... Staphylococcus aureus Cas9. Nature 520:186–191 . https://doi.org/10.1038/nature14299 Find plasmids from...targeting with CRISPR–Cas13. Nature 550:280–284 . https://doi.org/10.1038/nature24049 Find plasmids from this...altered PAM specificities. Nature 523:481–485 . https://doi.org/10.1038/nature14592 Find plasmids from this...and high DNA specificity. Nature 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX,...Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) NGRRT or NGRRN Neisseria meningitidis...than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9...more about SaCas9 and AAV Check out our CRISPR Featured Topic Page Resources on Addgene.org Check out...

...preparations are added to cells in culture. Three to five days later, cell cultures are inspected for sterility...also performed by ddPCR.  Quality Control Addgene ensures high quality viral vectors by optimizing and standardizing...quantity of gram-negative bacterial endotoxin is ensured to be less than five endotoxin units per mL. The...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified...item (see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... darker than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation...packaging system developed by the Trono lab. Cell culture medium (OptiPro SFM) containing lentivirus is first...

...samples: What we did To measure the pH of our samples, we used a solution of cell culture medium, which is buffered...samples at room temperature and used the color of the liquid solution and pH paper to measure the pH. We compared...range of pH exposures. They showed that AAV infectivity was not altered by a short 2-hour exposure to pH as...-rings are, in fact, permeable to CO2 at low temperatures (-80°C) and once in the tube, the CO2 can alter...not all O-rings) were permeable to CO2 at low temperatures. We tested two different types of tubes: the...tubes after the indicated incubation periods, and measured the pH. We found that tubes subjected to a recovery... 2014). They also showed that even 24 hours of exposure to pH 5.1 did not alter the infectivity of the...

...pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common pull-down...using antibodies against the target protein. The captured protein complexes are then analysed by mass spectrometry...contains the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started...protein to form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which...using standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments...RNA (IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly...vectors or synthesize/purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags...

... the off-target activity. Figure 2: Specificity is the measure of off-target activity (left) while...sensitivity. Site accessibility Site accessibility is a measure of which genomic sequences can be targeted by the... sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...closely related to specificity. Sensitivity is a measure of the on-target activity of the Cas9 nuclease....frequency at a target site. A specificity ratio can be measured by calculating the on-target activity divided ...while sensitivity is the measure of on-target activity (right). Cas9 nucleases that exhibit enhanced...NGAN, NGNG, NGAG, and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9...

... adjustments I wanted to make to ensure smooth filming in the future, but we were finally refining the...I could be happy with it. Cycle 5 - The Future! Figure 5: The mobile monitor and teleprompter being...trying all over again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for...anticipated, we had to think of something else.   Figure 2: Ironing boards. Good as a WFH desk, and as a...important for me to let people record at their leisure. For virtual B-Roll (that’s the secondary footage...drawing board.  Cycle 3: A Return to HQ Figure 3: Even Blugene is practicing social distancing... left the room. Microphone: Lastly, I needed to figure out a microphone situation. Generally, I record...

...extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction (or...overlap ensures that the PCR generates a nicked circle rather than a linear product (see figure). Template... simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust the extension time to ...vivo to give the final circular plasmid product pictured here. B) Screening of recovered plasmids for the...final concentration). The DMSO reduces secondary structures of the DNA template, and may also decrease the...template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through...polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary bases...

...Staphylococcus aureus Cas9. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr...some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello G... mouse model, an important proof of concept for future preclinical and clinical work. Find Plasmids for... is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and AAV ...homology-directed repair (HDR) pathway is downregulated in mature tissues - no problem, as exon skipping proceeds...would be applicable to 80% of DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular...low-frequency indels will not cause frameshifts. Figure adapted from Osterout et al. (2015b) under a CC-BY...

... like pipets, or mini-centrifuges, etc. Be sure to capture as many of these tools as possible, this even... we suggest estimating the time it will take to secure your first (R-level) research grant.  Set your ...down prices as you create your list. This helps to ensure that all of the resources are accounted for. Write...an idea of how much you can expect to use in the future. 3. Identify the skills needed to use these tools...the talent pool that you’ll need to tap for your future research team. For example, if you’re going to ...help you define salary costs. 4. Ascribe a cost structure associated to these tools, skills, and personnel...searching for a faculty position. This is important to ensure that you’re looking at institutes that have the...

...action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can ... which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial...implantation of fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains...improved photocurrent properties: The sequence and structures that determine membrane expression and plasma... electrophysiology, a technique that’s used to measure electric current of ion channels in living cells...electrophysiology. All thirty were functional and their measured photocurrent amplitude, wavelength sensitivity...were then characterized further by expression in cultured neurons and mouse brain slices collected following...

... Design and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), ...Interactions between the target strand and the gRNA (see figure below) Because off-target sequences have less ...theoretically decrease off-target cutting. Figure 1: Strand separation and target DNA binding to ...improve the specificity of Cas9 derived from S. aureus. This means you can likely apply similar rational...of genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers..., Chen et al. 2017 used single molecule FRET measurements to determine which portions of Cas9 might be...HypaCas9 plasmids are available from Addgene here! Future possibilities for Cas proteins With their enhanced...

...bacterial culture into the late stationary phase. Growing the culture at a lower temperature such as 30...  Figure 3. Summary of plasmid multimerization troubleshooting tips. Portions of this figure were ... More research is ongoing to determine plasmid features that affect the frequency of multimerization. .... Theoretically, as long as all the plasmid features remain intact, plasmid multimers function just as...recA-independent mechanisms. Avoid overgrowing the bacterial culture Another way to prevent plasmid multimerization ...optimizing the incubation time can help maintain cultures in log phase growth. In a study by Williams et...plasmid can exist in a bacterial population as a mixture of monomers and various forms of multimers (Summers...

...p300-P2A-PuroR for lentiviral expression. Figure 1: dCas9-p300 adds H3K27ac marks to histones in...plasmid form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters...also available as ready-to-use lentivirus. Figure 3: dCas9-DNMT3A methylates cytosines at promoters...LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC...silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted ...period, as this mark was faithfully propagated in culture and in vivo. In certain cases, epigenetic modifiers...acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038...

...experiments require positive and negative controls to ensure proper interpretation of results. A positive control... to compare the experimental group.     Figure 1: A, Most biological experiments will benefit ...group and C, technical replicates or repeated measurements will increase the power of the results. In this...experiment. Technical replicates are repeated measurements of the same sample and are included to assess...long as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should...molecules through the interactions of other antibody structures, such as the Fc region. Fc-mediated binding interactions...as with an isotype-matched control.      Figure 3: A preimmune serum control addresses situations...

...polysaccharide structures, many of which are easily varied by the cell and whose structures are just beginning...around 8-12 h at room temperature as opposed to overnight at optimal growth temperature. If you’re lucky enough...traction lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell... for by manipulating the nebulization time and pressure. Nebulization units, which can be purchased cheaply...attached with a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing...some clones being outcompeted by others in liquid culture, dilute and plate the complete library of transformants...paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to subject your membrane...