... The higher the titer, the better. AAV transport and transgene expression It’s clear that we need high...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... higher retrograde AAV titers for successful transduction. In another example, neuronal gene expression..., fewer channel proteins are required for the transduced cell to become light responsive than fluorescent...process of generating viral particles. Limit tube transfer - the fewer opportunities your viral particles...make sure to rinse off the surface of the filter membrane with the surrounding solution to ensure excess...

...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... 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...to focus on what you should be doing : writing a grant, thinking about the next project, reaching out to... transferable skills...

...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... turn your slide deck into a chalk talk. Your translator doesn’t know the term “diving board” so you end...remember to breathe!)   Public speaking as a transferable skill The worst thing about doing public speaking...most difficult form, which makes it the perfect transferable skill. Almost every other form of presenting...Conflict Resolution Transferable Skills Guide: Creativity...

... blog Management for ScientistsTransferable Skills Guide: LeadershipTransferable Skills Guide: Teamwork...the right time. Like many of the posts in our Transferable Skills series, this post is not meant to be ... that you could strengthen. The right set of transferable skills, coupled with your technical skills, ... can help, and if and when you find yourself transferring this skill to another job, it will be important...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...willing and able to solve, don’t take that skill for granted. When you go on interviews or are looking for a...

...feel they lack formal training in a variety of transferable skills. At Addgene, we've set out to fill this... 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: Problem-Solving. I don’t know ...in my more recent positions outside of the lab. Granted, I currently work on the Scientific Support Team... careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined. In this ...your life. This tactic is a good way to see how transferable these skills really are, while also practicing...systematic, logical thinking.  If you are already a lab veteran at this point, you probably have better developed...

...express a transposase or reverse transcriptase so they can transpose. Both class I and II transposons can be...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence that moves... encodes the proteins the transposon needs for transposition. Transposition protein: This is the protein...themselves as RNA transcripts and then rely on a reverse transcriptase enzyme to reverse transcribe back into ...reverse transcriptase and integrase enzymes which are required for LTR retrotransposon transposition. Non-LTR...reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons...2021. To mobilize a transposon, first the transposase enzyme binds the transposon’s long terminal repeats...

...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... 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....

...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...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...readout of relative reporter translation. Single molecule continuous translation tracking TRICK allows you...multiple rounds of translation from this single mRNA; you get a view of translational dynamics at the single...

... are often found with cellular exosomes, small membrane vesicles or microvesicles (approx. 40 - 150 nm... diameter) that are secreted from the cellular membrane into the culture medium. AAV particles are either... vectors. They found that they have a higher transduction efficiency, i.e. the same number of AAV particles...to modify it to improve yields. For example, transfecting the producer cell line with an expression vector...proteins, nucleic acids) which possibly improve transduction properties. György and colleagues found that...that the association of AAV particles with membrane vesicles protects them from antibody recognition and...AAVs achieved so far? Exo-AAVs have improved transduction profiles in different AAV serotypes both in ...

...learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how..., R bodies do not exert force by dynamically rearranging into different polymerization states. Instead...with proteins and remain functional, 2) break membranes outside of their natural context or 3) be modified...demonstrates that R bodies can function to break membranes of different organisms. And finally, we demonstrated...could use them to lyse cells (and surrounding membranes) in keeping with their natural function (see below...Silver, P. A. A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synth. Biol...

...?  Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Tranfection...define plasmid host range, identify a few of the features that confer broad host range in plasmids in nature...features to create broad host range recombinant vectors.  A plasmid’s host range is determined by the different...plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow host range, while others...identified as broad host range plasmids. The properties that influence host range are not fully understood...genes necessary for transfer initiation between bacterial hosts. Also, self-transmissible plasmids may have...examples of a narrow host range plasmid with only one ori and a broad host range plasmid with multiple oris...

...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...

...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with...cell membrane isn’t powerful enough, what about altering the chemical composition of the membrane itself...SuperPLD hydrolyzes membrane PC to PA. A) OptoPLD is recruited to plasma membrane or lysosomes and its...optogenetic tools to control the cytoskeleton and cell membrane dynamics. First, a quick refresher.  The tools...undergoes a conformational change and binds the plasma membrane under blue light CRY2-CIB1 heterodimerizes or ... separate chromosomes during mitosis, support transport of organelles and vesicles, and more. Although...demonstrated its use to probe cell mechanics, vesicle transport, and cell division, but given the long list of...

...that they lack formal training in a variety of transferable skills. At Addgene we've set out to fill this... highlighting that researchers do learn MANY transferable skills while working in the lab and by offering...

...resources on the Addgene blog Read about other transferable skills Find all of our science career related...

...may not translate well to other cell types. To overcome this issue, try transducing with a range of dilutions...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... for getting the most out of your lentiviral transduction experiments. Love your cells and they'll love...culture practices can lead to low titers and poor transductions. Cell lines should be routinely subcultured ... cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting ...

... in a nonacademic direction, there are many “transferable” skills you have developed in addition to learning...your favor? Look for opportunities to practice transferable skills in ways that will also enhance your science... concrete things you can do to develop those transferable skills while you are also learning to be an ... transferable skills...

...improve your grant writing skills. Good luck! Further advice on grant writing Since successful grant writing... during the grant application process. When my PhD advisor first asked me to write a grant application... advice on writing a grant proposal. "Murder Most Foul: How Not to Kill a Grant Application" by Vid Mohan-Ram...their advisor alone applies for these competitive grant applications; however, in some cases, your advisor...advisor may choose to allot some if not all grant duties to trusted students. For example, if the proposal...thoughts such as “I have no idea how to write a grant!”, “I don’t have time for this if he wants data ...that first experience, I learned the details of grant writing, volunteered to take the lead on various...

...terminators and resistance cassettes for selection of transgenic plants. The complete GreenGate toolkit, now available...the time required to generate multi-construct transgenic plants. In the most practical terms, what GreenGate...Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013)...

...third Addgene/Seeding Labs plasmid grant and plans to use the grant to order plasmids she can use as positive...in PCR-based cervical cancer screens. This third grant represents the continuation of our efforts to provide...Bengyella and Kwabena Duedu who were awarded similar grants back in June, Gladys will be using plasmids in ...will be opening the Addgene/Seeding Labs Plasmid Grant to a third round of applicants at the start of 2017...Superhero 5k Find New Cancer Resources Learn About Our Grants to Rare Disease Research Resources on Addgene.... seeding labs plasmid grant...