...this useful. Now that we’ve been doing this for almost 6 months, you may be looking for more ways to make...don’t all get the mentoring support we need, and most of us could use more mentors to listen, guide and... virtual!   2. Practice public speaking For almost any science career, there will be a time when you... about trying your hand at a video presentation? Most video platforms make it easy to record yourself....audiences in your future.     3. Start something In almost every career talk I present, I’m asked what Addgene... different criteria for different roles but for almost every position, we look for candidates that have... interviews, for more details on how to make the most of these exploratory conversations. If you set yourself...

...sites on different chromosomes in living cells, the system requires high-copy chromosome-specific loci in...study the spatial and temporal arrangement of chromosomes and how these arrangements may affect nuclear...based multicolor labeling system to visualize chromosomal loci in human cells (Ma et al., 2015). To do ...inter-locus distances. They tested this system out on chromosome 19 by targeting 46 repetitive loci that contained... (PR1), the p-arm (T1), and the q-arm (T2) of chromosome 19. Image from Ma et al., 2018 with permission...Hanhui et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proceedings of the National...

...the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus...pyralis) and perhaps not surprisingly it is also the most commonly used bioluminescent reporter. This beetle...from the firefly beetle (Photinus pyralis) is the most frequently used bioluminescent reporter, other luciferases...substrate coelenterazine and is likely the second most commonly used luciferase. Other organisms from which...assay The typical luciferase-containing plasmid is most often used to investigate the effect of regulatory...Many luciferase assays require cell lysis as the most efficient means to disrupt the cell membrane and...

...which chromosome the transgene has integrated and make genetic crosses with balancer chromosomes to make...that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct comparisons...longer because you need to determine in which chromosome your transgene has integrated. This can be accomplished...accomplished with a few matings to balancer chromosome lines but is not necessary with targeted insertions...insertions (here you would know beforehand on which chromosome your docking site is located). This amounts to...genomic locus of integration is. The white gene is most often used, but other selection markers include ...

...There are a lot of viruses out there – most of which we would all like to steer clear of! However, a ...them a little better. Here we will discuss the four most commonly used lab viruses: gamma-retrovirus, lentivirus...host's genome and is used to make more virus. The most studied (and used in the lab) type of gamma-retrovirus... have a cargo capacity of ~ 8 kb, ample room for most experimental needs, and can transduce (infect) a...integration, and its association with disease. The most well-studied lentivirus is HIV, which many lentiviral...replicate episomally, essentially remaining an extrachromosomal part of the host genome that can replicate... However, the cargo capacity of AAV is ~4.7 kb, almost half that of lenti and gamma-retro viruses. This...

...your lab, don’t be surprised. They are present in most of the cell culture facilities, tissue culture labs...is difficult to be detected in the serum that is most commonly used for every cell culture. Reusing the...agar plates. This method is capable of detecting most of the mycoplasma species, apart from a few of them... are used. A broad range of primers encompasses almost all the infecting mycoplasma. The primers used ...Prevention of mycoplasma contamination The best and the most important ways to avoid mycoplasma are by providing.... Plasmocin is a widely used drug that can clear most of the mycoplasmas present in the culture media.... MRAs are non-toxic, effective in the removal of most of the mycoplasma contamination, easy to use, and...

...also has the flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA.  To use the lambda ....   This setup can be also be used to modify chromosomal genes. After the initial editing event, the defective... defective prophage can be removed from the chromosome of the host E. coli by a second lambda red recombination... system is best suited for editing bacterial chromosomal DNA because it is easy to remove the recombineering...responsible for excision of mini-λ from the host’s chromosome. After this, mini-λ can readily be purified from...PCR can be used to screen for positive clones in most cases, and restriction enzyme digest can be used... being targeted for modification: the E. coli chromosome, a plasmid, or a BAC.    Pro Tip: ssDNA substrates...

... skill in its most difficult form, which makes it the perfect transferable skill. Almost every other form...Public speaking is not just one of the most common phobias–it’s a key part of being a scientist and an... ideas clearly and strategically is powerful in almost any career. It allows you to share and build support...and take a deep breath before the panic sets in. Most of the time, that’s enough–but if it’s not, I can...memorable and awful. So try to keep some perspective. Most people won't notice, or won't remember and those...is that you will have to learn this skill in its most difficult form. You’re presenting complex data, ...

... if the mutations are on the same chromosome. Homologous chromosomes recombine during meiosis, creating...homologous chromosomes in the parental generation, they are likely to stay on separate chromosomes in future...function when hemizygous, meaning that only one chromosome needs to contain the transgene in order to see...distance between the two loci of interest on the chromosome, the less likely crossing over is to occur between... which would get both mutations onto the same chromosome. Until crossing over occurs between the two loci...

... types of viruses generated in the lab. The four most common types of viruses grown in the lab are gamma...infection process (though it can integrate at chromosome 19q13.4 qtr. (AAVS1)). For these reasons, AAV...to work with BSL-2 materials, such as lentivirus. Most of this work will need to be done in a biosafety...a significant risk for aerosol contamination. As most centrifugation has to be done outside of a biosafety... and scrapes from sharp equipment are one of the most common forms of lab accidents, so extra precaution...significantly reduce the risk of an accident (the most common reason for lab accidents is not following...corners. For many researchers, viruses might be the most dangerous things they work with, and that can understandably...

...region of chromosome 15. They both result from the inactivation of genes on one copy of chromosome 15 (the...be ‘read’ by the cellular machinery. This is how most cells in the body contain the same DNA but have ...histone proteins that compacts cellular DNA into chromosomes and reduces the risk of dangerous DNA damage....the addition of methyl- groups to the DNA, which mostly occurs at CpG islands (regions of the DNA with ...syndrome). In both cases, the other copy of the chromosome is normally imprinted and silenced, and so the.... In both diseases, genes from both copies of chromosome 15 are inactivated, but different phenotypes ...

...techniques Intravenous Intravenous (IV) injections are most commonly administered through the tail vein — many... are given between the skin and the muscles. The most common injection sites are flat and easily accessible...the coordination down. Subcutaneous injections are most often used when injecting a cell suspension to create...Intrathecal Intrathecal (IT) injections are probably the most advanced needle injection technique. Intrathecal...expert No-needle techniques The term “injection” is most often associated with needles, but it may surprise...tricky given their small size. This technique is most often used for viral delivery, though drug delivery...can target the airways before the lungs. This is most often used for delivery of viral vectors, though...

..., Addgene had almost 18,000 CRISPR plasmids in our repository, and we have shared almost 300,000 CRISPR...plasmids. That’s almost a ninety-fold increase in size! From this collection, we’ve shared almost 300,000 plasmids... nucleases, the original CRISPR tool, remain the most popular deposited and distributed CRISPR plasmids...Addgene were knockout libraries, and they remain the most popular with our requestors. However, we can again...seem to never truly go out of style. Instead, an almost even mix of plasmids (by age) are requested and...

...FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to another protein...used to study many biological questions. The two most common uses are: 1) to test the expression level...shift in wavelength from excitation to emission. For most FPs, Stokes shift is less than 50nm (often much ...environmental changes (e.g. pH, Ca2+ flux, etc). The most commonly used are GECIs - genetically encoded calcium...distinguish between closely related colors. Furthermore, most FPs  have a broad range of emission which will be... (EGFP) was optimized for 37°C, and is therefore most suited for mammalian or bacteria studies, whereas...or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a few seconds to a few minutes. Photostability...

...choose to move away from academic science; there is most likely abundant opportunity to continue such discussions...discussing recent publications in a specific field(s). Most anyone who has spent any time in academia has likely...meeting takes place to ensure success. One of the most important is letting people know it’s happening.... be tailored to the group and format but should almost universally include the questions “Would you attend...discussed topic, and level of general busy-ness will most likely the primary drivers of frequency. At Addgene...start with one per month. When to call it a day The most ineffective type of meeting is the one that no one... should be well planned and carefully organized. Most importantly, they should be regularly assessed for...

...thinking about what comes next. To that end, one of the most important things that you can do now is diversify...landing my own position! Conferences One of the single most important things you can do to plan for your career...working professionals, with the added benefit that most people at the fair will probably be willing to accept... it stand out with key words and by putting your most relevant experience at the top. You may also consider...Here you can search for jobs, post your CV, and, most importantly, manage your professional network. This...hope to have, you must think outside of the box (most likely your lab, in this case). It is important ...Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014-3 https://www.forbes.com/sites...

...and reference ladders are not next to empty wells. Most people will simply load an extra ladder or technical...proteins after transfer. Troubleshooting The two most common (non-technical) issues with transferring ...different formulations instead of concentrations. Most labs use protein-based buffers, so here’s a quick...take time for you to get it to work well. Dilutions Most antibodies come with a recommended dilution and/...antibody incubation temperature and time. Though the most common conditions are 4 °C overnight or room temperature...(TBS) or phosphate-buffered saline (PBS) are the most commonly used solutions for washing. If your background... The last step in a western blot tends to be the most straightforward. While there are some things you...

...loading control will almost certainly be overloaded. One paper called this problem “the most common error associated...This is a job for a semi-quantitative western blot. Most western blots benefit from a positive control — ...know is present in every lane of your gel. At their most basic, loading controls help you rule out errors... linear detection range. As a result, they have almost the same value on the band intensity axis. This...The loading control can be accurately quantified Most housekeeping proteins are highly expressed in cells...slope indicates higher sensitivity, linearity is the most important feature of a loading control, and both...

...applications - now let’s dive right in! You know qPCR is most commonly used to measure relative quantification...correctly is imperative. Picking your chemistry  The two most common qPCR chemistries are hydrolysis probes (which...of pipettes and pipetting methods to find what’s most consistent in your hands.  When using multichannels.... They are often included in commercial sets and most machines have a protocol that automatically performs...performs the normalization step.  Machines Most general-use protocols, unless otherwise specified, were ...number of methods available for analyzing qPCR data, most with plug-and-play Excel spreadsheets available ... your analysis method. ΔΔCT/CQ method One of the most commonly used methods is the ΔΔCT/CQ method, which...

...However, when I enquired about roles in this field, most jobs needed at least 3 months experience working... practicalities of working in the field with the most basic equipment and was a tremendously positive ... that all commercial companies are like this, as most have a more stable funding situation and level of... of work than research or academia, and so most positions in industry are more stable than this. However...experience is that the technology is so advanced that most of the procedures in the lab are done with robots... the phone or explain how various plasmids work. Most people who call or email me are scientists who are...self-motivated, because you need to work independently most of the time and plan your own work schedule.  I ...