... The destination vector contains sites with complementary overhangs that direct assembly of the final ...Unique 4 base overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled...fragment in an inverse orientation. This allows for multiple DNA components (promoters, genes, terminators,... reaction. For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury...editing which allowed for the ordered assembly of multiple DNA fragments to create TAL effector nucleases...plasmid, but it can also be adapted to express multiple gRNAs. Addgene has two Golden Gate-based gRNA ... 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages of Golden Gate cloning...

... SCF E3 ubiquitin ligase complex. In the presence of auxin, this TIR1 complex binds to the degron sequence...elegant system that allows for the assembly of multiple DNA fragments efficiently and accurately in a ...a nucleus -> DNA compaction around histones -> Examples of histone-modifying enzymes and reader-effector...assembly (backbones available at Addgene) with examples of potential synthetic chromatin mediators and...system will allow investigators to rapidly create multiple synthetic chromatin-modifying proteins quickly...protein degradation and quantification of this depletion using imaging-based measurements (Ashley et al...of your protein interest would also result in depletion of the FP, which can be easily visualized.  The...

...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out...possibly be made up of. The answer is… drum roll please… non-coding RNAs! You probably didn’t see that ... However, removing large lncRNA fragments with multiple gRNAs poses the risk of disrupting adjacent genes...transcription of the ncRNA (Hazan and Bester 2021). For example, inserting a polyadenylation signal downstream ...interactions between ncRNAs and adjacent genes are complex and not yet fully understood, so it is important...is not effective for your experiments, there are plenty of other CRISPR-based options that can target ncRNAs...variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave single-stranded ...

...optogenetic tool called PdCO.  This is an "optoGPCR" (coupled to Go, so it's an inhibitory opsin!) and due to...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV_EF1a-DIO-PdCO-mScarlet-ER-miniWPRE...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV_hSyn-PdCO-EGFP-WPRE AAV1, AAV5...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV-CaMKIIa(0.4)-eOPN3-mScarlet-WPRE...AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV-CAG-FLEX-rc [Jaws-KGC-tdTomato-ER2...AAV1, AAV9 Biosensors Li New viral service with multiple serotypes pAAV-hSyn-GRAB_OTmut AAV9 Biosensors... AAV9, PHP.eB Controls Feng New viral service, multiple serotypes Looking for more viral vectors? ...

...Level-1). Other possible tools, lentiviruses for example, are not approved for use in vivo use at the University...Manitoba due to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves are...you can see, AAVs of each serotype can transduce multiple tissues. So how do you choose the right serotype...hippocampus and cortex) within the mouse brain (4). An example from the cortex is outlined in the table below....thawing. Although more stable than other viruses, multiple freeze thaw cycles can still reduce AAV titer....is possible to use AAV constructs with self-complementing structure (scAAV) (7). As single stranded DNA...mapping of circuits In our lab, we use AAV-DREADD complexes to study neural function. Often the reporter proteins...

...techniques differ in important ways, but can also complement one another by providing both broad and specific...Mismatch Cleavage Assay Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing...investigator can be relatively certain that their samples don’t have off-target mutations if they remain ...the clinic. NGS-based methods provide the most complete information profile regarding putative off-target...counts making them fast and viable options for multiplex sequencing in many laboratories. Nevertheless,...sensitive as targeted amplicon sequencing. For example, GUIDE-seq seems to have a minimum detection limit...DSBs, doesn't require high read counts, fast multiplexing Requires delivery of dsODN (potentially toxic...

...amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple, and gfasPurple) (Figure 2). Figure 2: Chromoprotein...expressed and gives the cell a magenta (amilCP) or purple (tsPurple) color. Though the toxin is integrated in ...). Eight years later, scientists isolated pink, purple, and blue chromoproteins isolated from reef-building...just by looking at the cells on the plate. For example, if your plasmid contains a gene encoding a blue... Figure 3: pTOX plasmids expressing amilCP and tsPurple genes. Image: Lazarus et al., 2019. One such... lab is the Waldor lab who used the amilCP and tsPurple chromoproteins to create negative selection markers... and Tse2 toxins, respectively, and encode the tsPurple chromoprotein.  Find the allelic exchange vectors...

...relatively easily synthesized, must be produced using complex mixtures derived from cells or using live cells... expression of mammalian proteins that require multiple post-translation modifications for proper protein...systems Insect cells can also be used to produce complex eukaryotic proteins with the correct post-translational.... These systems enable high expression of very complex, glycosylated proteins that cannot be produced ...expression. Protein expression in bacteria is quite simple; DNA coding for your protein of interest is inserted...pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein expression from each of these vectors ... expression systems, including the majority of complex post-translational modifications. Extraction and...

... CRISPR samples. We invite you to jump in and try it out, or take a look at our live examples: experiment...adhere to the same principles listed here. It employs biological best practices. For example, it ranks guides...November, we began using it internally. Guiding principles behind CrispyCrunch While CrispyCrunch may evolve...you can depend on it to stick to a few guiding principles that we've followed since the beginning. It'...if desired. It works as a whole or in part. For example, you can analyze experiments designed outside CrispyCrunch...active guide. With these constraints in mind, we implemented the following algorithm for optimal mutation ...Francisco. Before starting his career in software, he completed a MSc in Psychology at McMaster University in ...

...some simple solutions available for researchers to deal with all of these problems. The simplest and best...Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many...forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment...cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely... a cell. Unfortunately, things are not quite so simple. Concentration is the number of molecules within...around that fusion protein is very high. Real world examples of these situations in cells are described below...order oligomers. Unfortunately, FP dimerization coupled with POI dimerization can lead to polymerization...

...create and simple to multiplex. Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters...easy to multiplex. Their design allows flexible swapping of fluorescent proteins and multiplexing is simple...reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with...properties that limit which fluorophores can be multiplexed together. Challenging  to Design: FRET reporter...

...briefly boiling the sample before use or by directly adding a small volume of the sample to the PCR reaction...orientation-specific primers. Blunt end cloning is an example of when you might want to know the orientation ... insert and one primer anneals to the insert. A simple way to create this type of primer pair is to mix-and-match...  Primer Design Pros Cons Insert-Specific Simple "yes or no" test results Doesn't tell you the... few ways you can do this. If you are going to complete your colony PCR analysis in the same day, you ... product size on a gel   Now that your PCR is complete, it’s time to run the products on an agarose gel...and to add a loading dye with glycerol to your samples before pipetting them onto the gel. The figure ...

...ever like to write about a recent plasmid deposit please sign up here.  Here's what you'll find in this ... list of specific structures within cells. For example, to improve nuclear localization of fluorescent...fluorescent proteins, they found that adding a triple nuclear localization signal or including fusions with Histone...//doi.org/10.1101/160374 Nanobodies- there's a simple method for that! By Michelle Cronin The benefits...Kimiko Hara, and Kazuo Umemura have described a simple method which alleviates the inclusion body problem...problem and makes nanobody production in E.coli as simple as following a DNA miniprep protocol!  First, the...https://doi.org/10.1016/j.pep.2020.105607 A new multiplexed CRISPRi and CRISPRa system for human pluripotent...

...needed to transform the raw sequencing data into a complete plasmid sequence that would be ready for our QC...inherent in Nextflow’s design, we were free to use multiple programming languages (Python, Perl, Java, R, ... adaptable property gave us the freedom to use multiple open-source sequence assembly tools at the appropriate...containers are they are designed to ‘contain,’ in a complete package, all of the pieces of software that depend...bioinformatics workflow in Nextflow Establishing a complete pipeline however, required more than just identifying...from the first process to the second process. For example, our bbduk process created trimmed FASTQ files ...sure we understood the concept of channels and implemented the input declaration correctly, the script block...

...lab often tests samples of new bacteria from anywhere they can find, whether the samples are from a rain...was safe to say the sample had their attention! They isolated DNA from the sample, amplified the CRISPR...phylogenetically classified as the more complex Class I systems that contain multiple subunits, but the more they ...Cas7-11 appeared to have derived from the more complex type III-A and III-B systems. Though it was composed...the domains into a single protein, shedding the complexity of its ancestors. They classified the system ...cleaving RNA at positions defined by the target:spacer duplex without detectable non-specific activity. Its major...

...appropriate time—for example, the cell must have enough nutrients available to complete the entire round ...and regulation in bacteria Before looking at an example plasmid, let's review how E. coli normally replicate... must bind ATP. Then, the ATP-bound DnaA binds multiple specific sites within the ori called DnaA boxes...stringently controlled plasmids, replication is tightly coupled to the bacterial host's cell cycle, maintaining... stringently regulate plasmid replication. For example, the plasmid ColIb-P9 encodes the protein RepZ ...Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot...replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process...

...process is simple -  no restriction, ligation or gel purification steps are required! Multiple fragment...highly efficient way to move DNA sequences into multiple vector systems. With the appropriate entry and... understand the process, we’ll walk through an example experiment where we might use Gateway cloning to...attL-entry vector. This fragment is inserted in a multiple cloning site (MCS) of an attL-containing entry...lentiviral expression, we could use a vector like pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible... cloning You can use Gateway cloning to insert multiple DNA fragments into many vectors at once in the...plasmids available at Addgene, including the Frew Lab Multiple Lentiviral Expression Systems (MuLE) Kit, the ...

... models. In complex diseases, it can aid in generation of multiple edits or implementation of expression...change in pathology. If the disease pathology is complex, a model may be most effective. Once drug candidates... all diseases are fully characterized or have a simple pathology. In these cases, preliminary identification... – the dreaded ‘undruggable’ proteins – having multiple hits or an entire pathway of possibilities greatly...made this control step extremely accessible. For example, let’s say you identified gene target X for BRCA-deficient...candidates are selected.      CRISPR aids in multiple stages of the drug discovery pipeline.   ...

...gene. For example, they eliminate the expression of the gene (by CRISPR knockout, shRNA depletion, etc.) ...genes – if a gene functions at multiple stages in development, its depletion may result in lethality before...redundant genes – multiple genes which function in overlapping roles can mask the depletion of one another...modifying the gene of interest in any way. For example, if green fluorescent protein (GFP) was the reporter...monitors due to transcriptional fusion.   Examples of promoter, enhancer, and gene traps at a genomic...

...slowly becoming a convert to the principle that you can't motivate people to do things, you can only demotivate...Intelligence or to hone delegation skills, for example. So aside from a focus on removing obstacles, what...grow in skills. Managing a team is hard and most people have some learning they need to do before they ...most from your team. It is pretty clear that if people are mostly happy at work, they will work harder...problems to solve (I think this is true of most people, not just scientists) Are self-directed, but need... in a 5 part series on Introduction to Managing People for Scientists. Subscribe to the Addgene Blog to.... Good introductory reference books on managing people: Skills for New Managers by Morey Stettner Successful...