...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...

...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...

... 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)...

...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...

...?  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...

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

...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...

...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 ...

...and Harvard Medical School. Scientists making transgenic organisms with Cas9 should be aware of the potential...conducted with extreme caution. Why does making transgenic organisms with Cas9 risk creating a gene drive...drives that can spread through populations of transgenic laboratory organisms but not wild organisms. ...targeting a synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both...collective technologies are developed safely and transparently.  For more information, visit www.sculptingevolution.org...

...The goal of Kiran Musunuru's lab in Harvard's Department of Stem Cell and Regenerative Biology is to ...modeling work in human pluripotent stem cells: a transcription activator-like effector nuclease (TALEN) kit... designer proteins that will introduce a double-strand break in the genome anywhere you would like. Cells...cells after the plasmids have been introduced by transfection or electroporation. All you do to create a protein...genetic backgrounds. It becomes more of an apples-to-oranges comparison. Addgene: Can you compare the TALEN ...the target site. Also, any time you create double-strand breaks, there is a possibility of introducing a...

...insoluble granules. We overcame this problem by engineering a plasmid which contained cold tolerant chaperones...bacteria out of their optimally suited temperature ranges and make them grow better at suboptimal ones, which...bitter cold.  Since our objective is to induce tolerance towards cold, we decided to focus on a special... of Earth’s harshest environments: corrosive pH ranges, crushing pressures, near-melting temperatures,... host cells (the simplest example would be a transformed mesophilic, for example E. coli, carrying a plasmid...hybrid system where we could incorporate cold-tolerance inducing genes within a regular mesophilic host.... And thus was born ELSA - our happy lil’ cold-tolerant E. coli! Additionally, we’re also working towards...

...CRISPR-associated Transposase) by the Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems...DNA transposons have been hindered by their limited programmability: depending on the transposases involved...-guided transposition. By natively combining the DNA integration capabilities of transposases and the ...crRNA.  Three transposase proteins (TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains... each type of CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE.... Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor molecule...characteristic of the Tn7-family of transposons. Insertion of the mini-transposon can occur in two different orientations...

...repeats (ITRs) are what makes an AAV transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases...read through this challenging sequence. Transgene The transgene is the gene that will be delivered by the...Most AAV transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid ...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...(WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which ... nuclear export of RNA and RNA translation, and promotes RNA transcript longevity. The polyA is located...proteins needed for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today...

...sybodies against membrane proteins Developing antibodies against proteins embedded in membranes is tricky because...develop antibodies against unstable targets like membrane proteins that may unfold due to camelid high body...synthetic nanobodies, called sybodies, against membrane protein targets in as little as 3 weeks (Zimmermann...lab can quickly select for sybodies targeting a membrane protein of interest using the Sybody Generation...have a concave, loop, or convex scaffold and are randomized with a defined set of different trinucleotides...serines and threonines at positions that can be randomized for creating the libraries (Zimmermann et al.... the process for selection of sybodies against membrane proteins. Image from Zimmermann et al., 2018. ...

...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...

...type VI-D enzymes are the latest tools in the transcriptome engineering toolbox. How were Cas13d enzymes...small hairpin RNA interference, dCas9-mediated transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA...only time will tell how large the genome and transcriptome engineering toolbox will be. It is, however,...community. References Konermann, Silvana, et al. “Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR...Editing with REPAIR Get tips for using Cas9 Transcriptional Activators Learn about Cas13a (C2c2) Characterization...