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...constitutive high level expression? Finally, you can add bells and whistles to your construct. If you ...specific antibody against the protein available – you can always add an epitope tag or a fusion protein. Mammalian...visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected ...selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade your basic mammalian expression system...containing pDEST-HisMBP). After purification the protein can be released from the resin and used for various binding...Consider your applications and experimental design. You can create a new strain with a disrupted gene by transforming...under the Gal4 inducible UAS promoter. Other vectors can be expressed in fly culture cells like S2 and carry...

...which can “drag and drop” kilobases’ worth of DNA with CRISPR-driven precision into a genome, can be considered...collateral" activity of the enzyme can lead to off targets and result in significant, negative effects in RNA targeting...forward for what the field can do.    References and Resources References  Özcan A, Krajeski R, Ioannidi...tests samples of new bacteria from anywhere they can find, whether the samples are from a rain gutter ... instance, a disease like cystic fibrosis, which can be caused by thousands of different mutations, all...lab switched up the serine integrase for one which can enzymatically take a genetic sequence and integrate...cargo via Cas9-directed integrases.   Use PASTE can be delivered via a single dose of plasmids, works...

...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...

... language is the basis of shuttle vectors, which can function in unrelated host species, even from completely... Figure 1: Shuttle vectors are vectors that can function in more than one host species. Created with...plasmids? If we all use the same molecular code, can I transfect any old bacterial plasmid into HeLa cells...Agrobacterium tumefaciens, a plant-infectious bacteria that can incorporate foreign DNA into plant genomes. Eukaryotes... wall synthesis. Eukaryotes, with no cell walls, can grow alongside these antibiotics just fine. This ...function. A puromycin resistance gene on your vector can protect both plasmid-carrying bacteria and eukaryotes...auxotrophic cells die. Some auxotrophic selection markers can be used across species, for example, in both S. cerevisiae...

...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...

... and binds cells that can begin an immune response. This important biomarker can be used to distinguish...Addgene's Cancer Collection Pages Learn How the ClonTracer Library Can be Used to Study Cancer Drug Resistance...types of cancer have different challenges and obstacles to overcome, mesothelioma sufferers can see great...inflammation, which can cause mesothelial cells to mutate and ultimately form cancerous tumors.  This process...researchers are hopeful that they can come up with new ways to discover the cancer earlier, in particular before...different avenues that can be used to treat it. For those who are running out of hope with more traditional treatments...is breathed in (or less frequently, ingested), it can get into the lining of the lungs or travel to one...

...issues as a region of interest can be targeted using a gRNA, which can easily be cloned into a gRNA expression... as dCas9 (Jinek et al., 2012). Like Cas9, dCas9 can bind to a specific DNA sequence via a targeting gRNA...DNA-binding molecule, such as Zinc-finger or TAL proteins, can be used. The Fujii lab demonstrated this in mammalian...proteins targeting a specific genomic locus, which can require multiple cloning steps (Fujita et al., 2013...identification of DNA binding proteins, this method can characterize nucleic acid-nucleic acid interactions...successfully used in this "in vitro" form. Any tag system can be used to isolate genomic regions of interest with...biotin ligase. Biotinylated dCas9, with bound DNA, can then be purified by streptavidin high-affinity purification...

... 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 ...

...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...

...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...

... 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...

...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...

...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...