...sample.  How do I make the standards for a standard curve? You create a standard curve by serially diluting...use qPCR with a standard curve to titer their viruses; food allergy labs use standard curves with ELISAs...them in their assays.  What is a standard curve? Scientists use standard curves to determine the concentration...diluting a known control sample, called a standard. The ideal standard curve has at least five dilutions, with... measurements. You'll need to prepare the standard, standard dilutions, and unknown sample(s) in the same...or volumes too large for a standard micropipette (> 1,000 µL). The standard dilution series should be ... value. How do I plot and use a standard curve? To create a standard curve, you graph the measured response...

...expression stable regardless of copy number (Segall-Shapiro et al., 2018). Designing stabilized promoters...keep gene expression stable regardless of copy number, the team made use of stabilized promoters. In contrast...and keeping its expression level constant. In the case of the stabilized promoters from Segall-Shapiro et...gene expression also remained stable regardless of plasmid copy number. Stable gene expression whether growing... were expressed. What’s the use of stable gene expression? Constant gene expression can enable more confidence... to unregulated promoters that are always on, stabilized promoters incorporate feedback loops such as ...with copy numbers ranging from 3-100 per cell, a stabilized promoter buffered against the effects of copy...

...tested at the beginning. Variability source Standardization parameters Animal Strain, age, sex, housing...conditions (individually versus in groups), health status, diet, cage enrichment, stress level, origin (vendor...Alternatively, for larger brain areas, repeated staggered injections may be considered. Timing: High expression...situations such as learning and memory or emotional states, for example anxiety or depression. In upcoming...

...choose to start their own academic research laboratory. How does one even go about starting a lab? Obviously...students and postdocs when starting out. She advises “do not hurry to staff up the lab and to take things...resources for hiring research staff. And for a more indepth resource for starting your academic lab, download...a PI without a personal assistant has to deal with. My inbox currently stands at about 65,000 messages...for advice on staffing your lab. Let us know if you have any relevant tips for starting a lab. Or share...effective working environment? Tom Ellis was offered a start-up package that included a postdoc and a PhD student...important to me to be with them in the lab when they started, so that we could discuss our projects and ideas...

... you. Statistics – The basics Statistical analysis is what determines if there is statistical significance... several core factors of statistics relevant for many downstream statistical tests.   Independent variables...are statistically different. They are classic parametric tests and one of the simplest statistical tests...described above. Violated assumptions and statistics Statistical power, the ability to correctly identify...is a better option. Stay tuned for our upcoming blog on non-parametric statistical tests! Adding it all...your friendly neighborhood biostatistician happy by making your statistical analysis plan when you design...Wissing, Dennis R., and Timm, Donna. Statistics for the nonstatistician: Part I. South Med J., 105(3): 126...

...Biomedical research, for instance, contributes heavily to medical practice, and understanding the scientific process...can vary "Lab assistants" usually wash glassware, prep media, etc. "Research assistants" are more involved... maintenance Administrative assistant: assists PI with admin duties Staff scientist: similar role to a...professors, and other mentors, and four years after I started college, I will be entering a PhD program to continue... As scientists, we probe beyond the realm of established theory. Realizing that textbooks don’t tell us... process can help us understand how new therapies can be developed and applied. The current pandemic serves...the lab Official title is typically Professor, Assistant Professor, Associate Professor Sometimes teaches...

...involved, it can be difficult for scientists to understand how GPCR activation leads to downstream effects... the endogenous ligand or drug. In the inactive state, the luminescent chimeric protein Gα-Rluc8 and Gγ-GFP2...resulting in a decreased GFP2 emission. The activation state can be measured by a BRET2 ratio, which is the ratio...pharmaceutical drugs, there is still only a partial understanding of how GPCRs signal through G protein signal...provides scientists with a toolkit to gain an understanding of the signaling through the transducerome from...as well as the activation by drugs to further understand their mechanism. These plasmids are available... the transducerome, to gain a more complete understanding of a GPCR mediated response.  Acknowledgements...

... creation of the United States Environmental Protection Agency, which started making regulations such ... the second in a series of posts on bringing sustainable practices in the lab. The first post focused ...learn about some creative approaches to lab sustainability, and find ways to further reduce our consumption...consumption beyond recycling.     Choosing more sustainable products Recycling is great for rerouting waste... grant agencies), we can shift toward a more sustainable one. The math on this is pretty simple: wash ...solutions and practices to meet higher environmental standards. Find some easy action items on our other article...

...location where the United States Postal Service picks up mail. NEB actually started this program in 1976! ..., flip the flaps of the box so the pre-paid postage stamp is on the outside of the container, and drop...off the box at any location where the United States Postal Service picks up mail. Recycle plastic packaging...more lab sustainability tips, check out our other articles in this series: Bringing Sustainable Practices...popularity of single-use items, plastic is a substantial portion of lab waste. Despite the fact that researchers...cost of regular waste services. Learn about how to start a glove recycling program at your institute! What...culture room for the wrappers and ship them back. Start a glove recycling program at your institute! Universities...

...was GeoCas9 discovered? The discovery of GeoCas9 started with an interesting question: why engineer a new...GeoCas9’s editing activity in HEK293’s. GeoCas9 stability in human plasma The last trick GeoCas9 has up ...Harrington et al suspected GeoCas9 might have higher stability since prior research had shown that a cocaine ...cocaine esterase engineered to have increased thermostability also had a longer lifetime in human blood. To ...lower than optimal temperatures. GeoCas9’s thermostability could also be used with in vitro molecular ...aspect of GeoCas9 are you most excited about? Thermostability? Longevity in human plasma? Something we haven...CRISPRing! References Harrington, Lucas, et al. "A thermostable Cas9 with increased lifetime in human plasma...

...can use the loss of resistance as a marker for successful cloning. In these instances, the cloning vector...Resistance to antibiotics is a widely used tool in molecular biology, yet scientists rarely stop to think...to maintain the plasmid. Adding an antibiotic resistance gene to the plasmid solves both problems at once...pressure to keep your plasmid. Viva la (bacterial) resistance!  What are antibiotics? Antibiotics are generally...bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction between the two ...categories may be a bit of a gray area as some bacteriostatic reagents can kill bacteria when used at high...subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin B polypeptide...

...Though Cas9 is not a static molecule in any binding state, RNA loading stabilizes the closed conformation...pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state (PDB ID 4CMP) with domains...the PAM is so flexible in this state that it doesn’t appear in the crystal structure (Figure 2) at all. ...Pro tip! X-ray crystallography cannot capture flexible or disordered regions, so crystal structures like...in this region. The crystal structure in Figure 3 shows the guide RNA stabilizing the DNA-binding protein...pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared to the guide RNA-bound state (PDB ID 4ZT0, bottom...engineering, and studying its structure can help us understand the parameters of CRISPR experimental design....

...cells, are already resistant to this antibiotic due to stable integration of resistance genes in their genomes...selection agent. Pros: Stable (spectinomycin). Cons: Cost (spectinomycin) or stability (streptomycin), doesn...damage. Choosing an antibiotic resistance Choosing the best antibiotic resistance ultimately comes down to ...antibiotic and happy selecting!   Antibiotic Resistance Stability Time Saving?  Works for multiple organisms... M., & Larminat, F. (2005). Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene...Plasmids need antibiotic resistance genes so that you can specifically isolate bacteria containing your...interest. But does it matter which antibiotic resistance gene you select? In this blog, we’ll break down...

...to compare the photostability of different fluorescent proteins? Measuring photostability Since it is clear... to quantify the photostability of fluorescent proteins. To determine photostability, an experiment is...Measuring photostability at realistic powers will provide better insight into the photostability in the ...Measuring photostability in living cells provides a much more realistic view of photostability. Selecting...This avoids substantial changes in excitation power and will help to keep the photostability of the probes...this series, we will discuss how to select a photostable fluorescent protein. Photobleaching is the irreversible...desirable to have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins...

...concept. It is imperative that we all understand what antibiotic resistance is and how it develops so that we...Microbiology Pages What is antibiotic resistance? Antibiotic resistance is defined as microbial survival despite...generally organisms with multi-drug resistance are resistant to three or more different classes, or types...Learn How Antibiotic Resistance Genes Are Used in the Lab How does antibiotic resistance develop? There are...in which bacteria can become resistant to antibacterial drugs. Resistance can be intrinsic, meaning a ...means the bacterium is not naturally resistant but has become resistant somehow overtime. One fantastic example... example of intrinsic resistance would be Mycoplasma species’ resistance to beta-lactam antibiotics, like...

...challenging for researchers using CRISPR, and that standardizing and improving the gRNA design process and rules...applications is necessary going forward. The standardization of CRISPR methods was a common thread at the...NIH Common Fund, emphasizes that we need more standardized methods for evaluating CRISPR technologies, ...basic or early pre-clinical research. However, standard terms and techniques are not enough to move the...including a detailed look at how cut-to-mutation distance affects editing efficiency. Optimizing Donor DNA...members of Jacob Corn’s lab describe how a better understanding of Cas9 biophysics can help enhance HDR. CRISPR...

... camera, OR, monitor from that distance.  Build: We got a wheeled stand and mounted a monitor to it. It...Addgene’s Science Video Production Specialist, I’m constantly thinking about ways to improve our productions...Pivot Back in March 2020, just four months after I started with Addgene, I was about to have a check-in with...resources we have, and we developed a great plan to start consistently knocking out high-quality videos for...the window. So, much like SynBio, it was time to start our Design-Build-Test-Learn cycles. If you’re not...filming myself. While easiest from a production standpoint, it wasn’t ideal for our Lab Protocol videos....space to shoot was another hurdle. As we slowly started to realize that we were going to be out of the ...

...musician, then metastasize is touring. But you know what a touring musician needs? A backstage team. Meet ...progression, and metastasis. The microenvironment’s physiology is deeply connected to these 3 stages and contains...microenvironments have started to gather attention. By the 1960’s, scientists have begun to understand two main areas... seem to take center stage in cancer development, the microenvironment backstage play just as important...Meet the microenvironment, a tumor’s backstage team. Microenvironments are cellular regions with specialized...highly prone to affect it. While tumors may be the stars, there's plenty of action outside of tumor cells...them, making it even harder to cure cancer. To understand how cancer cells influence microenvironments ...

... Marc Zimmer’s recent book, The State of Science: What the Future Holds and the Scientists Making It ...their inclusion here adds much needed context to understand every other problem. For non-scientists, Zimmer... ago. Teaching by example No examination of the state of science would be complete without the inclusion...to. The problems and challenges described in The State of Science have not changed in “COVID times,” but...next week.   A stepping stone When I discussed The State of Science with the five Addgene colleagues who ... learn everything about one specific topic, The State of Science could be a jumping off point to explore...

...number of participants and the standard academic calendar. So you want to start a journal club? Here are some...begin building a high level understanding across multiple teams or to stay abreast of current trends in...certainly had concerns that I may not have time to stay current or enjoy that discussion. Luckily, one needn't... edge science, researchers of all varieties must stay current with literature and emerging technologies...recurring meeting amongst scientists that aims to help stay current on new and emerging work by collaboratively... The first thing to do if you’ve thought about starting a journal club is to find a topic and seeing if...clear purpose and effective time management, for starters.  Now, you are ready for your first journal club...