...they perform complex biological tasks, both of which are factors in the technical challenges they can ...hail Marys that have helped me troubleshoot some technical obstacles when working with virus: 1. Viral vectors...Different titering methods can reveal differences in physical versus functional titer. If you have low titers...cells, replace the medium with half the volume typically used (e.g., use 5 mL medium on a 10 cm plate)....increase transduction efficiency. These reagents typically enhance the adsorption of a virus particle to ...measured with branched-DNA technology." Journal of clinical microbiology 37.6 (1999): 1683-1686. PubMed PMID... virus adsorption on target cell membranes." Biophysical chemistry 97.2 (2002): 159-172. PubMed PMID: ...

...cable is threaded through—and fixed to—an optical insulator called a ferrule (below). The fiber optic cable...maintained during the entire protocol, otherwise optical stimulation will be erratic, opsin activation/inactivation...stimulation parameters must also be determined empirically but it’s best to try those that have already ...flexibility in behavioral experiments. However, the technical demands of using these wireless devices are significantly... in cerebellar Purkinje cells. The Journal of biological chemistry. 2011;286(29):25848-58. doi: 10.1074... Sparta DR, et al. Construction of implantable optical fibers for long-term optogenetic manipulation of...PMC3466483. 4. Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless optogenetics...

...must make is which system you will use, or more specifically, the type of cells that will be used to make...spanning both prokaryotic and eukaryotic organisms. Typically, what system is used is dictated by the specific... one passage (transfection). This differs from typical insect cell preparations, which we talk about below... below. Processing The viral supernatant is typically collected over the course of a couple days to increase...supernatant will depend on the virus type. AAV is typically purified using methods like ultracentrifugation...produce baculovirus, a family of viruses that specifically infect insects. This is what makes insect cells...transfer plasmid and the baculovirus construct, called a bacmid. Like AAV and lentiviral packaging plasmids...

...smaller libraries, chemical transformations can also be used. Efficiency for chemical transformations is...expected transformation efficiency, or more specifically calculated based on the size of and weight of your...that of electroporation, but an advantage is that chemical transformations are generally cheaper and easier...easier. One library that Addgene distributes with a chemical transformation amplification protocol is the Chong... KB) Screening Rinehart Lab Iterative Synthetically Phosphorylated Isomers (iSPI) Libraries ~11,000...DOCX, 17 KB)   For CRISPR libraries, Genome-scale CRISPR-Cas9 knockout and transcriptional activation...

...to your normal control - you can set up your calculations so that your assay output number (RQ value) ... In that case, an RQ value of one (or, more realistically, between 0.80-1.20) would mean you have one ... on and so forth. This also serves as a light technical control - an RQ value of 1.5 means you have either...incredible discovery. The range of RQ values used to call copy numbers should be as small as reasonably possible...all double-stranded DNA, while Taqman uses an intercalating dye, with a fluorescent probe and quencher to...). Sybr, therefore, cannot be used to accurately call copy numbers, though it has been shown to identify...all researchers. Accuracy needed Do you need to call exact copy numbers or just general trends? How confident...

...Kusumika (Kushi) Mukherjee, editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. Stem...differentiate into cells of peripheral blood. Typically, there is a very small number of ASCs in each ...Limited. Only specific cell types can be produced Ethical concern High. Embryos are destroyed during ESC ...of post-transplantation rejection when used therapeutically High Low (if ASCs are isolated from the patient...bypass the transplantation compatibility issues and ethical concerns of ESCs. As iPSCs can be sourced directly...directly from a patient’s adult cells when used therapeutically, the chances of post-transplantation rejection...Kushi) Mukherjee is the Editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. She...

...graduate student culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We ...public. My experiences in grad school, though often called “nontraditional,” helped prepare me to start CINSO...educating the public and organizing events. I contacted local professional organizations and nonprofits. I volunteered...nontraditional” career path long ago. Use your analytical mind to test and discover what career suits you...mistake, I evaluated what lessons I had learned – call it “troubleshooting.” Trying out new experiments...followers. It’s also been a great place to reach out to local establishments such as museums, professional organizations...State Museum and others when I was trying to find local informal science educators, and ever since, the ...

...MCC develops and makes freely available technical and pedagogical resources, offers mentoring for instructors...best teachers and mentors pass on more than just technical knowledge, shaping their trainees’ very way of...their research capabilities and laboratory and critical thinking skills (Knutson et al., 2010a,b). Classroom-based...at other institutions to form a support network called the MDH CUREs Community (MCC) that seeks to reduce...student enthusiasm, and research backs up that view (Callahan et al., 2022).     Fig. 2: Students in...Chapter 17, Einstein Discovers America. New York./  Callahan, K., Peterson, C., et al. 2022. External collaboration...classroom-based undergraduate research experiences spanning chemical biology, biochemistry, and neurobiology. CBE Life...

...Produce Chemically and Functionally Distinct Vectors. Molecular Therapy - Methods and Clinical Development...and community concerns AAV vectors are complex biological reagents; many factors, like storage buffers ...out of fear it points to flaws or gaps in their technical ability. But, especially for AAV data, that may...experimental settings. It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER...Hub. These minimum data standards are designed specifically to allow scientists to evaluate how small changes...evaluate AAV vector performance. Yet, most of the technical data needed to replicate and build on others’ ...word of mouth” as their source for this type of technical know-how, which highlights the dearth of AAV vector...

...Balancing research with medical practice as an MD/PhD. Darrel does 20% clinical work 80% research and was...for Regenerative Medicine at Boston University Medical School. Darrell’s research focuses on the lungs...Center for Regenerative Medicine, and within the biomedical research community. Along these lines, Darrell...Pathology and Laboratory Medicine. He attends in the Medical Intensive Care Unit, the Pulmonary Consultation...the Pulmonary outpatient clinic, all at Boston Medical Center. In addition he conducts basic stem cell...Regenerative Medicine of Boston University and Boston Medical Center. Special thanks to our sound engineer/producer...

...Within that research group, you will also typically receive technical mentorship from a ‘bench mentor’. In ...receive after you’ve found your mentor, though. Typically, you will identify a ‘scientific mentor’: someone...scientific project. Your scientific mentor is typically a professor who operates a research group. Within...component. Documenting during training It’s hard to recall a lot of details, especially when you are in a ...to do? Walk through the protocol mentally (or physically where relevant) and identify any gaps or questions...bring them to your mentor. Pro tip! Consider physically walking through the protocol, by going to each...experiments. Unlike in lab courses, where everything is magically made up by helpful TAs or assistants, and sitting...

...have been used since the 1930’s in a range of biological studies, including investigations into environmental... are powerful resources for investigating any biological process. High quality genome sequence A high-... mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish...thousands of mutations in genes affecting diverse biological processes—not just development—are available ... zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes, morpholinos...One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the...Naturally, the best model organism depends on the biological question, and zebrafish are not ideal for every...

... Predoctoral Fellow at University of Rochester Medical Center. During development, complex genetic programs...central nervous system (CNS). To address this technological gap, a trio of recent papers describe the development...Cre under neuron-specific promoters, including calmodulin-dependent kinase II alpha (aCaMKII; excitatory...with different untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional...demonstrated by the Qi Lab, which developed a subgenome-scale CRISPRa screening approach in embryonic stem (ES...Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently a Ruth... Predoctoral Fellow at University of Rochester Medical Center. He studies gene-function relationships ...

...often scarce and difficult to identify. So what specifically should you do? What follows are just a few suggestions... in fact, some of the very things that were so critical to me landing my own position! Conferences One...Society for Cell Biology (ASCB) or the American Chemical Society (ACS) are just 2 examples of organizations...will sometimes result in a recruiter giving you a call. Many companies enlist the assistance of recruiters...appropriate job candidates. If you do receive such a call, make sure to get that person’s contact information... get the specific position he or she originally called about, you can keep in touch and reach back out...know all in one place! This information can be critical to a focused and effective job search. As with...

... the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC)...have tended to avoid cell culture in favour of biochemical assays followed by in vivo testing. The mantra...Estimates of the amount of money wasted in basic oncological research due to contamination are in the tens...prevented and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach Scientist...experience through working for a variety of pharmaceutical companies including GSK, AstraZeneca-MedImmune...talks and seminars on cell culture and other pharmaceutical topics. The LGC Group also provides standards...Learn about the Dangers of Using GFP for Protein Localization Resources on Addgene.org Find Plasmids for...

...for neuronal/CNS expression. Modulated by calcium and calmodulin. GAL1, 10 General expression mRNA Yeast... promoters only work in prokaryotic cells and typically only in the same or closely related species from... be ways to turn them on or off at will with a chemical, heat, or light. In the cell, promoters themselves...scientists have created synthetic promoters, which typically include some combination of other promoter elements...Inducible with Tetracyline or its derivatives.  Typically contains a minimal promoter with low basal activity...CaMKIIa Gene expression for optogenetics mRNA  Ca2+/calmodulin-dependent protein kinase II promoter Specific...phosphage dehydrogenase Constitutive  Very strong, also called TDH3 or GAPDH. ADH1 General expression mRNA Yeast...

... request for phages to the directory does not typically come out until nearly the last minute when all...all other options have been exhausted. Theoretically, phage alerts can be sent out to the directory earlier...team must decide which labs should receive the clinical sample to test their phages. Because the sample...phages targeting organisms closely related to the clinical sample. For treatment, it is best to identify ... only used phage in basic research but not therapeutically: not all labs in Phage Directory have the resources...resources or know what it takes to prepare phages for clinical use. There are also different regulations from... individual phages. Integrating lab protocols, clinical information, phage research job postings, and ...

...Chinese Academy of Medical Sciences University of Texas at Austin University of California, San Francisco  ...scientific community support open sharing. But, one critical part was missing when the COVID-19 plasmids first...Children's Hospital                           California Institute of Technology             Johns Hopkins...                           National Institute for Physiological Sciences Broad Institute                                       ...part of research projects, another way is to systematically offer thousands of constructs for tagged and...enables research on solute carriers (SLCs) for medical research Recently, the Michael Davidson Fluorescent...

...also more precise. Base editors, for example, typically edit all the target bases within the base editing... target nucleotides are positioned within the canonical base editing window, base editing has higher efficiency...products to be indels while the efficiency of HDR is typically less than 10%. In contrast, the original paper...maximum efficiency isn’t everything — prime editing typically needs to be optimized for each application, and...nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which had prime editing efficiencies...optimized for human codons; additional nuclear localization sequences; and two mutations in Cas9 previously... 3′ end, the Liu lab appended RNA pseudoknots, calling this longer RNA an “engineered pegRNA” or epegRNA...

...GFP is tagged with a nuclear localization sequence, allowing it to localize to a different area of the ...mOrange2, EGFP, and mKate2 (red) to solve the problematically low fluorescent intensity of previous versions.... XFPs are introduced using a plasmid construct called Zebrabow-GateDest containing an RFP, CFP, and YFP...developed a version of Tetbow that incorporates chemical tags instead of XFP to optimize it for tissue ...architecture of a fluorescent neuron is visible. Chemical tags are more resistant to tissue clearing methods...neuronal circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed PMID: 30454553...homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells." Cell 143.1 (2010)...