...spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, while maintaining... brought to the target location using a gRNA and can then recruit Cas3, which carries out the brunt of...abundant type in the Class 1 category. These type Is can then be further broken into subtypes of A–G, which...appears to be conserved. This DNA degradation portion can be bypassed by leaving out Cas3 — a tactic some CRISPR..., developing Cas3 tools has been challenging. It can be difficult to ensure comparable expression of all...efficiency of these systems, though consistent expression can still be a challenge. A multitasking Cas Most Cas...pulled through Cas3. After these initial steps, Cas3 can perform its helicase and nuclease functions simultaneously...

...plasmid DNA sample can be treated with RNase to remove RNA contamination and you can use a phenol chloroform... filter. Each glass filter can capture up to 150 ug of DNA, and filters can be stacked together in series... equipment. Plus they create plastic waste. Kits can also have the annoying tendency to runout right when...hours to complete, while this syringe-based method can be completed in 20 to 30 minutes. Additionally, while... purposes, with the Kim and Morrison method, you can just adjust the number of glass filters you use depending...cellulose directly into a PCR buffer, where DNA or RNA can be amplified. Compared to a commercial magnetic bead-based...yields as fresh columns, suggesting that columns can be reused at least five times. DNA purification kits...

...Additionally, an AAV reference, a virus with a known titer, can be used to confirm that the standard curve is giving...for ddPCR. One issue with qPCR is that the results can vary by a factor of 2. This means that if you set...found that generating the standard curve for qPCR can be challenging to get right. Here at Addgene, we ...technology. One well (containing thousands of droplets) can be sufficient to capture the information needed for... concentration of the sample. This concentration can then be used to calculate the viral titer: GC/mL ...should only need to use one. However, two channels can be used to examine the integrity of the viral genome...control (NTC) well is common. Here are some steps you can take to achieve a clean NTC: Have a dedicated bench...

... tips and tricks you can use to obtain optimum transformation efficiency. It can take 6-8 weeks from the...Tsuyoshi Nakagawa lab are binary gateway vectors and can be used for promoter swapping and generating N and...your constructs are ready for transformation, you can cut off the primary inflorescence (the flowering ...seeds that you know won’t harbor your transgene. You can optionally remove them to reduce the number of non-transformed...strain. Additionally, the transformation process can get a bit messy, so you want to make sure you have... another 24 hours. The remaining starter culture can be used for colony PCR and glycerol stocks. Dip/drip... insertions occurred. Having too many insertions can cause artifacts from over-expression and it is likely...

...two additional parameters that can affect FRET efficiency. These issues can be overcome by choosing a complementary...coupling." The emission of the acceptor fluorophore can be measured using microscopy techniques. FRET measurement...fluorophores and their orientation to one another can affect FRET. When it comes to studying an unknown...measure FRET. The table below lists plasmids that can be used to create your choice of fluorescent fusion...fused between ECFP and Venus Other issues that can affect FRET measurements include: the brightness ...pair too close to each other in the spectrum, you can easily directly excite the acceptor with the laser...the FRET methodology for a given experiment. You can then choose the most efficient approach for your ...

...decide to share them using Addgene’s repository, you can also share your associated protocols. When you upload.... And more plasmid-related educational resources can be found in our Plasmids 101 blog series. Want to... Addgene? Check out all the benefits and how you can get started on our deposit page. Protocols.io Website...a life science protocol repository. The software can be used with the ZappyLabs Bench Tools mobile app...to use, and add your own comments. Currently you can find protocols from NEB, OpenWetWare (see below),...allows labs to create accounts in which their members can share protcols, sign-up for shared equipment, and...software designed with scientists workflow in mind and can offer a one-stop solution to your lab's organization...

...hosted new posts on topics ranging from new tools you can use to monitor cellular perturbations to how CRISPR...These properties, combined with other benefits you can find in the post, may make Cpf1 the preferred CRISPR...editing this post, I learned a lot about how FRET can be used to study biological phenomena and about how...how fluorophores other than fluorescent proteins can be incredibly useful in FRET experiments. Professor...recommend this post to anyone wondering if and how they can apply FRET in their own research. All posts from ...

...guidelines to follow in order to ensure that people can quickly and easily identify what your plasmid contains...your plasmid name. This simple piece of information can often convey many important details. Once you know...know the backbone a plasmid is based on, you can usually derive: a) the bacterial antibiotic resistance...scientist to know what is in a plasmid and often how it can be used just by reading the name. For more information...information about plasmid features and tips you can use in the lab, check out more posts in our Plasmids 101...

...Indeed, this method can be used at high throughput because numerous cell lines can be generated, each ...the pull-down assay, wherein a protein of interest can be captured along with its associated partners. Common...biochemical “epitope tag” such that these tagged proteins can be immunoprecipitated using a standard tag-specific...interest, and a universal all-purpose “tag antibody” can be used for downstream IP/ChIP assays. Creating ...epitope tagging. Using our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency across...Integrated DNA Technologies, USA). These synthetic RNAs can also be purchased from various other suppliers with... it is also straightforward as the RNA molecules can be directly purchased and there is no need to construct...

...(Martella et al., 2017). If you can dream up an experiment, MoClo can help you achieve it! More recently...constructs for a variety of applications. MoClo tools can be used in bacterial and cell-free expression systems...IIS restriction enzymes (Weber et al., 2011). It can be performed at multiple levels to generate anything...variety of purposes beyond what we discuss. If you can think it, MoClo might help you achieve it! MoClo...The EcoFlex Kit has a wide array of DNA parts that can be assembled into individual TUs or very large (up...offers a one-pot reaction wherein multi-part assembly can occur. It is quite extensive with a supplemental ...special attention to protein modifications which can be especially difficult in alternative expression...

... proximity of the two proteins can be inferred. Intermolecular FRET can be experimentally difficult to...push these tools even further? All of these things can be done using FRET! Read on to find out more about...dipole-dipole interaction. The efficiency of FRET can be measured for a given donor-acceptor pair and a...consideration for a FRET experiment, the possibilities can seem overwhelming given the large assortment of fluorescent...proteins are available on our FRET Resource page and can be used to create fusions with a gene of interest...efficiency and any unpaired fluorescent proteins can contribute additional noise to the measurement. If...FRET, well-characterized FRET reference standards can be used to validate FRET measurements and serve as...

...extrachromosomal arrays can lead to artifacts due to overexpression. CRISPR can insert the FP gene into...they are great in number, generally very small, and can contain different neurotransmitters. Using genetically... synapses or that are Cre-inducible, researchers can explore synaptic integration and connectivity without... large genetic changes in C. elegans with CRISPR can be difficult since it requires the much less efficient...single-stranded oligodeoxynucleotide donors (ssODN). So how can you use CRISPR to introduce large FP genes into C...mouse embryonic fibroblasts. This fusion protein can be used to determine cell cycle length by examining...Addgene CasRx is a RNA targeting Cas system that can be used in Drosophila melanogaster. Get the plasmids...

...an initial reversal drive cannot restore the exact original sequence, it can restore the original protein-coding...distort inheritance in their favor and consequently can spread even when costly. If even a single organism...appropriate flanking homology into a germline cell can create an RNA-guided gene drive. In principle, this...combination with standard barrier protocols, they can reduce the risk of accidental escape to a negligible...Molecular confinement involves building gene drives that can spread through populations of transgenic laboratory...to human error. And with gene drives, one mistake can be enough. Reversal drives are designed to overwrite...sequence using a recoding strategy; a subsequent drive can restore the wild-type sequence (save for the residual...

... soon as you can. It is in your supervisor’s interest that you succeed, and if you cannot resolve a problem...on their promises, Wet-blanket negativist, who can bring down the morale of a group with their pessimism...to check in with you?” For a specific project you can ask when he or she would like an update, and whether...problem on your own, reach out for help so you can get the support you need. 2) Prepare an agenda for ...immediately get your supervisor’s attention so you can focus on the problems that need to be resolved. If...more about your work than your supervisor does. You can save yourself and your supervisor some time if you...your supervisor, you need to demonstrate that they can count on you to follow through on your commitments...

...getting feedback from the team. Successful management can almost be boiled down to one, key concept: Creating...conversations and gestures are energetic.  This can take practice and training. This is important because...with the team leader. There is no way a manager can be efficient without delegating and empowering the...information flow so everyone knows what is going on and can contribute. Members periodically break, go exploring...effective team regularly seeks expertise wherever it can be found. Listen to our podcast interview with...anonymous surveys with regular frequency. Anonymity can be a great way to get honest feedback, but shouldn't...meetings with team members (don’t become known for cancelling these); ask for feedback and input on multiple...

...probes. You can follow him on twitter: @joachimgoedhart.   Marieke Mastop is a PhD candidate at the Section...This is time well spent and, if you do it right, can be (part of) figure 1 of your next paper or thesis...best fluorescent protein for a specific application can seem like a daunting task. Many fluorescent proteins...expressed at an equal level (Goedhart et al, 2011) can be used. An assay developed in our lab uses a reference...conditions, a ranking of the practical brightness can be made (Goedhart et al, 2012, Bindels et al, 2017...fluorescent protein, a ranking of practical brightness can be made. This strategy has been used in yeast by ...practical brightness provides a better picture of what can be expected in a 'real' application than the theoretical...

...type target in each sample can be quickly calculated (details in [2]) and can be used to quantify rates...many thousands of partitions, statistical modelling can be used to determine the actual number of target ...abundance, does not require standard curves, and can theoretically detect a single target molecule in ...digital PCR works, let’s explain what kind of assays can be used to sniff out your desired mutations. The ...assays often used in real time PCR. These assays can be designed to detect either a donor sequence integrated...product. Low detection limits: Digital PCR assays can accurately quantify low levels of mutant target (... of the plethora of available sequencing options can be found here. Good luck with your genome editing...

...Secondly, fluorescence imaging of living organisms can be challenging due to issues with tissue autofluorescence...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and...so that they are parallel to each other and thus can no longer fit with β1-9. They connected β10-11 with...DEVD). Upon cleavage of the sequence, β10 and β11 can rearrange to their normal antiparallel position and...proof of concept that the designed used in FlipGFP can be applied to other fluorescent proteins to create...demonstrated that the FlipGFP-based caspase reporter can be used to visualize apoptosis of enterocytes in ...die or cells dying at the wrong time and/or place can lead to an array of diseases and pathologies such...

...endogenous RNAi pathways. These mutations however can interfere with other genetic pathways making it difficult...proteins within minutes, but requires H2O2, which can be toxic in living cells. BioID was derived from ...for labeling, but the process is much slower and can take as long as several days. In 2015, the laboratory...stomach flu and food-borne illnesses, noroviruses can infect millions annually. Because it is highly contagious...solution: a low-cost, cell-free detection assay that can be completed without special equipment. What’s more...detected through a toehold switch, an RNA sensor that can be used to detect the presence of any specific RNA...specific to a particular strain of norovirus, it can be tailored for the detection of other norovirus ...

...multiple secondary antibodies can bind to a single primary antibody, this approach can greatly increase the sensitivity...that a complete Western blot can be run in a single day, I really cannot recommend it except in the most...isotype subclasses, such as mouse IgG2 or IgG4. This can be important for some applications. Assay use While...conjugated in the lab. In the indirect approach, you can simply keep a stock of conjugated secondary antibodies...specific isotype subclasses of primary antibodies can increase the possible number of target proteins.   ...method. But in reality, multiple primary antibodies can and do attach to a single target protein, so this...analysis of a sample. Instead, relative quantification can be done using either the direct or the indirect method...