...apply to programs, and apply for travel grants. Good luck and happy travels! Resources Resources on the ...remote collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork...current institute. A common reason is hands-on training in a technique that only a few labs have expertise...this expertise will provide you with guidance and training so that you yourself can become an expert and ...for another reason. Visiting would allow you to train and acquire the data you need on the new instrumentation...new collaborations and fresh perspectives. For trainee level researchers, it can expose them to a new ... Identify reagents you will need, any required training, and a priority list of experiments you hope to...

..., exo-AAV2 in mouse retina after intravitreal injection to transduce bipolar cells and photoreceptors ...22314290. PubMed Central PMCID: PMC3345986. Meliani, Amine, et al. "Enhanced liver gene transfer and evasion...PubMed PMID: 29296848. PubMed Central PMCID: PMC5728288. Schiller, Lara Timantra, et al. "Enhanced Production... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...particles is able to infect more cells (as demonstrated in different cellular environments such as retina...also found that exo-AAVs were more resistant to neutralizing anti-AAV antibodies. This is particularly useful...vivo where endogenous anti-AAV antibodies often neutralize the therapeutic efficacy of AAV vectors. How ...

...resources on the Addgene blog Transferable Skills Guide: Time Management Transferable Skills Guide: Conflict ...of being a scientist and an incredibly useful transferable skill. Being able to stand up in front of an... audience and convey your ideas clearly and strategically is powerful in almost any career. It allows ... applies: what can go wrong, will go wrong. The trash removal service slams dumpsters outside the open... turn your slide deck into a chalk talk. Your translator doesn’t know the term “diving board” so you end...messing up?–and use the three tips above to make a strategy that can help. Even something as simple as telling...remember to breathe!)   Public speaking as a transferable skill The worst thing about doing public speaking...

... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high... the issue of low titer by concentrating your virus. See our Concentrating AAV Protocol (Step 10) for ...PubMed Central PMCID: PMC2574934. 3. Zingg, Brian, et al. "AAV-mediated anterograde transsynaptic tagging...contact with the cell is to increase the AAV concentration. In this flowchart, you can see the dwindling...be required. For example, retrograde AAV gets transported up the cell’s axon. While the mechanism for this...yet fully understood, we can imagine that this transport is probably not 100% efficient. So the few AAV...particles that have entered the cell now have to travel up the axon, through the cell, and potentially ...

...researchers feel that they lack formal training in a variety of transferable skills. At Addgene we've set out... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: managing a team. You’ve just been...about management skills during your scientific training. I never thought about management skills before...grad school. Both were hard-working scientists-in-training who got the job done, but I struggled with what... he graduated. Years later I attended a formal training for new managers and I had a “Eureka” moment while...around the fact that new team members need to be trained, which may seem like a waste of your time at first...

...researchers feel that they lack formal training in a variety of transferable skills. At Addgene we've set out... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...where you might need some help. Today in our transferable skills guide: Time Mangement for Scientists....to-do lists at the end of the day so they can jump straight into tasks the next day. If you work in a job ...useful to avoid such procrastination: Remove distractions: Probably the easiest way to do this is to leave...internet and all it’s enticements are far too distracting. (As a side note, I’d recommend the book, “Bored...more fantastic reasons to remove yourself from distractions every once in awhile.) Reframe the task in a...

...have been used to track protein localization, cell structure, intracellular trafficking, and protein turnover...with SapTrap The Jorgensen Lab recently developed a modular plasmid assembly toolkit called SapTrap to ...selection marker for screening modified strains. With SapTrap, the user first designs either oligos or...fission/fusion, nuclear import, and neuronal trafficking. Although FPs have enabled discovery of many ...of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a genomic site specified by a guide...challenging to screen for the genetically modified strains that contain your newly tagged gene. Most insertions... improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one tube assembly...

...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...of relative reporter translation. Single molecule continuous translation tracking TRICK allows you to ...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...destination co- or post-translationally. TRICK: Visualizing the first round of translation While ribosome profiling...in the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary...to see the first round of translation, but it doesn’t allow continued tracking of a given mRNA because ...

...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...delivery is more common than conventional transgenic strategies. In such vector-based gene delivery systems... XFPs in the original method, we used the Tetracycline trans-activator system to boost the expression ...expression levels, and we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...troubleshooting your Tetbow experiment Plasmid concentrations and AAV titer - The XFP genes have to be introduced...highest color variations. Adjust the plasmid concentrations or AAV titer for the best results. The timing...

...DEAE-dextran can improve transduction efficiency. In addition, this study found that transduction efficiency...passage number of a culture the lower the transfection and transduction efficiencies. Cultures should only be...others opt for a single harvest after transfecting with the transfer and packaging plasmids. A benefit of...type may not translate well to other cell types. To overcome this issue, try transducing with a range ...Zhang, X.Y., Reiser, J.. "Production, concentration and titration of pseudotyped HIV-1-based lentiviral...vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events... for getting the most out of your lentiviral transduction experiments. Love your cells and they'll love...

...E. coli expression strains  Note: All strains are derived from the E. coli B strain, except ** which are...induction strain for example, nor will a p15 plasmid be compatible with a pLys strain. Your strain may require...popular commercial strains designed to avoid them. Why do I need an expression strain? Protein expression...below, certain specialized strains are available to confer greater transcriptional control, assist with proper...most expression strains to accommodate high protein levels including:  ompT: Strains harboring this mutation... These strains have an inactivated native restriction/methylation system. This means the strain can neither... K12 Strain Resistance Key Features Genotype Use BL21 (DE3)   Basic IPTG-inducible strain containing...

...emotions, such as frustration or anger, because your discussion will get side-tracked, and you might create... your project and work hours. While specific strategies to deal with each of these personality types ...these principles it would be “assertiveness.” 7 strategies to become more assertive & resolve conflicts ...supervisors are difficult people. The following seven strategies will help you to become more assertive and resolve...between you and your supervisor, you need to demonstrate that they can count on you to follow through ...professional conflicts. At the workplace, these strategies will help you to communicate with confidence ...your supervisor is a difficult person, the above strategies will still be helpful but you will need to be...

...the E. coli B strain. Common E. coli strains used in the lab Most of the commercial strains you find today... the intestinal tract of animals. There are many different naturally occurring strains of E. coli, some...commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain and the...the B strain. K-12 was isolated from a patient in 1920 and eventually led to the common lab strains MG1655...1918 but was first referred to as “B strain” in 1942. The BL21 strain and derivatives are the most common...changes found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...of the popular strains, their genotypes, and their primary use in the lab. These strains are all based ...

...with a 3-part strategy: Train: Nearly every new Addgene employee gets “Lexicon Training.” Since the lexicon...you can be incredibly frustrating. Clarifying the words —> decreasing frustration —> increasing the happiness...and racks meant we had fewer errors in our LIMS tracking system. Though you might not have to communicate... lexicon is a “living” document, I update the training frequently. In hindsight, getting a list of frequently...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

...an article describing a sucrose supplementation strategy to improve yield. In addition, as described in..., the team developed a viral genome sequencing strategy to confirm the identity and purity of all AAV ...! AAV purification by iodixanol gradient ultracentrifugation This video goes over AAV vector purification...protocol page for more detailed instructions. AAV titration by qPCR using SYBR green technology Here’s some...used to generate a standard curve, visit the AAV Titration by qPCR written protocol page. Getting started...

...competitive transplant assay in the mouse intestine, demonstrating its utility in long-term tracking in vivo...provides a combinatorial indexing strategy that simultaneously tracks clonal history and cell identity ...linking ancestral gene expression patterns with reprogramming outcomes (Biddy et al., 2018). Tracking lineage... tool that combines scRNA-seq and lineage tracking to track the dynamics of gene expression and clonal... of CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts...reprogrammed into iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like...reprogramming (CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), ...

...emission spectra for BV421 and BV711 on a 3 laser spectral flow cytometer   Using spectral flow When...autofluorescence extraction feature. Although autofluorescence extraction is an optional feature of spectral flow ...portion of that spectra to use for compensation.     Figure 1: The emission spectra and filters ...cytometer.   Spectral flow cytometry Full spectrum flow cytometry, or “spectral flow” cytometry, ... be distinguished using spectral flow since the machine uses the full spectral signature, not just a narrow...in the spectra can be used to distinguish these two fluorophores from one another during spectral unmixing...with spectral flow—you can run a three color panel or a 40 color panel! When preparing a spectral flow...

...Enhancer traps Like promoter traps, enhancer traps lack enhancer elements and require the trapping of endogenous...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems lack... enhancer traps! However, don’t count the other traps out just yet. Gene and promoter traps unambiguously... up on their modern counterparts. What are traps? Traps – gene, enhancer, and promoter – all rely on...in fly, mouse, and yeast models. Why traps are useful Traditional genetic screens employ various on/off...expression of the gene they monitor. Gene traps Gene traps are promotor-less constructs that contain ...gene product resulting in a transcriptional fusion, much like promoter traps. While this system does not...

...NEB’s new teaching space at Lab Central to learn all about it. LabCentral is a lab and office space shared...because both LabCentral and Addgene aim to make scientific advancement easier. NEBNext Ultra FS DNA Library... prior NEBNext Ultra II DNA Library Prep Kit for Illumina, one was only able to extract libraries from...New England Biolabs (NEB) hosted workshop at Lab Central and discuss why these types of workshops could ...in a lab.   Participating in a workshop at lab central We’re working on a project where we aim to do large...throughput DNA prepping by trying out NEB’s new NEBNext Ultra II FS DNA Library Prep Kit for Illumina sequencing...people to take a break and relax. Companies at LabCentral get access to a variety of experimental services...

...your AAV packaging transfection. In my lab we regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper...prevent future headaches. To help you plan PEI transfections for AAV packaging, considering using this PEI...Calculator. AAV packaging typically requires transfecting three plasmids at specific molar ratios. To ...PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging Select the plasmid...pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue culture dish ...dish you will be performing the transfection in: 15 cm dishes OR CF5 stacks Select how many dishes or ...or stacks you plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet: Name your...