...the game rules and then you let your students play. As your students play, you can observe, answer questions... make our classes more practical and hands-on. Students in these courses get plenty of theory in their... genetic manipulation of human stem cells when students are not even allowed to manipulate prokaryotic...cells? Especially, if you only have 4 hours and 35 students! After a course coordination meeting with some... I didn’t want to face another group of sleepy students at 8 a.m. many of them focused more on social ...therefore started planning a game that would force my students to use concepts learned from previous lectures...win-win strategy. After each team presented, all students had to pick the best strategy and propose a final...

...labor with up and coming immunotherapy treatments. As treatments enter the clinic, physicians and researchers...keep ahead of the disease in patients and developing collaborative treatments will go a long way in combating...macrophages (M1 and M2) that help create welcoming environments for tumor cells spreading to other organs during...to inhibit further cancer growth and subjecting patients to the risk of microbial infection. Chronic inflammation...one of the most commonly used immunotherapeutic agents. Currently the FDA has 12 approved monoclonal antibodies...cells and T cells activated ex vivo are also contestants for cell-based immunotherapy. Using activated... CRISPR-based clinical trial in 18 lung cancer patients. This study used CRISPR gene editing to knockout...

...microenvironment, a tumor’s backstage team. Microenvironments are cellular regions with specialized structures...structures. Tumors are surrounded by microenvironments and what encircles a disease is highly prone to affect...precisely that for breast cancer. What are tumor microenvironments made of?  A tumor’s genesis is divided into...development? Being pathological active regions, microenvironments shape a tumor’s evasiveness, evolution, and...others repress it. Unfortunately, as a whole, microenvironments promote tumor durability instead of repressing... To understand how cancer cells influence microenvironments and vice versa, let’s take a look at cancer...improving cancer cells proliferation.  How do microenvironments affect tumor spread and drug resistance? Although...

...from their contents. You should be tracking both, you should easily be able to move contents from one container...can be useful in academic labs where postdocs, students, and lab mates are coming and going frequently...Addgene website, it sets off a chain reaction of events throughout all of Addgene - within the office, ...applied to containers and not the mushy, gooey contents of said containers, think carefully about how ...encoded numbers. Loosely couple containers and contents Right from the get-go be as clear as you can be...are being applied to the containers, not to the contents. Tightly coupling barcodes and containers might...such a bad idea, tightly coupling them to their contents might leave you sad and confused when a tricky...

...1-3 kb (approximately gene-sized) fragments so that these fragments can be cloned into E. coli and subsequent... Express random phage DNA fragments in E. coli Now that random DNA fragments have been cloned into E. ...return no candidates. Our strategy: Express random fragments of the phage genome and probe with live cells ...DNA frequently enough to result in a smear of fragments that get smaller in size as a function of digestion... method that results in the bulk of resultant fragments falling between 1 and 3 kb results in optimal ... causing issues associated with cloning large fragments. After fragmentation, repair sheared ends with...into chemically competent E. coli and analyze transformants for appropriately sized inserts. We consider...

...fixation/permeabilization reagents for intracellular targets. Since knockout experiments can be time-consuming...selectivity of an antibody for flow cytometry experiments. This will include a discussion of the Human ...approach that prioritizes more reproducible/renewable reagents, such as recombinant or hybridoma-derived monoclonal...cells that are amenable to genetic knockout, this represents a very robust approach to antibody validation...staining. This can include using: different blocking reagents; titration of antibody concentration (this can... need to study protein expression at multiple timepoints. When the target of interest is critical to cell...increase confidence that the labeling observed represents the expression level of the target of interest...

... easily access the documents. These tools not only allow users to access documents from anywhere, but ... papers on your desk, and to tackle some new experiments before the next R01 is due. But what about organizing...re frustrated and no closer to starting your experiments. Get organized now! Start by gathering that pile... color coded binders and tabs (for different experiments or projects) to make it easier to know where ...go Never miss a deadline again. Organize you experiments, keep track of upcoming grant deadlines, and ...from webpage clippings, photographs, and file attachments. Organize your notes into notebooks, create checklists...required to change the status quo. But with recent improvements to ELN and online lab management software, there...

...selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA fragments, such as... as gene replacements and tags.  The typical frequency of recombinants is 1 positive clone out of 104 ...Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants. Proceedings of the National Academy...homology to the target site for recombination. Key components of the Lambda Red recombineering system The lambda... lambda red recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three...modification with an ssDNA substrate.  Gam: Gam prevents both the endogenous RecBCD and SbcCD nucleases...an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system. See ...

...cell to perform multiple recombination events, all linear fragments need to get into the same cell. For ...3), producing multiple fragments with specific homologous ends. These fragments are assembled in vivo ...multiple recombination events to assemble the final plasmid. At least 5 fragments can be correctly assembled...Figure 1). As this approach requires no special reagents or purification kits, it is low cost and can be...protocol:  Fragment generation - Any linear DNA fragments can be assembled in vivo as long as they have ...PCR is the primary method to produce the linear fragments, as desired modifications and homologous regions...positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid assembly...

...essential components of both pathways have been shown to be efficacious for increasing knock-in events (Yang...mutations and gene editing Targeted genome editing events fall into two general classes: knock-in and knock-out...frameshift mutation. The majority of cellular repair events will generate these types of mutations, making ...DNA repair mechanism in general. On average, it accounts for less than 10% of DNA repair, with this rate...considerations for designing donor DNA for successful HDR events. CRISPR cut site location Position your CRISPR...disrupting mutations in donor DNA   Promoting HDR events over other edits Like I said earlier, HDR is not... to exist as a repair template. To maximize HDR events, ensure cells are actively cycling through the ...

...of the vector components are HIV-based? A7: No single plasmid contains all the components necessary to ...The components are divided as follows: Transfer vectors contain minimal cis-acting HIV components: LTRs...specific experiments being performed and you should understand the guidelines for using such reagents as outlined... produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer ...Second-generation vectors are likely sufficient for most experiments; however, the 3rd-generation packaging systems...the 3'LTR in the transfer plasmid. The deletion prevents full-length viral RNA from being produced in target...LTRs, PPT, RRE, and psi packaging signal. Viral components typically total <1.5kb, which is rarely more ...

...NGCG 8  Tips for designing base editing experiments in plants Choose a base editing platform depending...engineering through adenine base editing in plants." Nature plants (2018): 1. PubMed PMID: 29867128. 11. Li...Plant base editor platforms and their molecular components. Adenine base editors for plant We developed... could be used to generate stable genome edited plants via Agrobacterium-mediated transformation. We also...). Type Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9...transient assay. For stable transformation, regenerated plants may be subjected to genotyping. If successful base... Yuan, et al. "Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A." Nature...

...result presents an opportunity for troubleshooting and optimization of the transfection reagents and process...There are many, many different types of experiments carried out by scientists every day. Although the... controls that can be used for plasmid-based experiments. What are control plasmids? Generally, control...interpretation of the results. Typically, plasmid-based experiments employ transfection, negative, positive, and ...control allows you to examine if the transfection reagents or the transfection process itself has any cytotoxic... on gene expression in your target cells. In experiments employing gene targeting or genome editing technologies...results are reproducible from properly controlled experiments. The observed phenotype associated with the independent...

...unique tropisms are now available. These include variants with broad CNS or PNS transduction, cell type ... control transgene expression. Gene regulatory elements [promoters and enhancers, miRNA target sites, ... 2021) to evolve new systemic capsids for both rodents and non-human primates (NHPs). These engineered...fluorescent proteins, optogenetic sensors, CRISPR elements, etc... *These are only starting recommendations...not been tested in rats. For more information on reagents and protocols, visit https://clover.caltech.edu...frequently been used for screening candidate regulatory elements such as enhancers and promoters for cell type-...Tabebordbar et al., 2021) method, selecting for variants that were effective in C57BL/6J mice, BALB/cJ ...

...selection easier, as well as highlight some recent advancements within this field. Fundamental principles of...enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation... function and its propensity to bind cellular components. Some dyes target specific organelles like mitochondria...may prove challenging to stain intracellular compartments in in vivo model systems, so extra care must..., highlighting its reliability for long-term experiments (Salari et al., 2019). Photostability, the ability...experiment types: Different types of fluorescent experiments, such as using fluorescently tagged ligands or...

... Identify reagents you will need, any required training, and a priority list of experiments you hope to...that they may want to consider for future work placements. The chance to learn and collaborate all while...which you can apply to. These structured types of placements often have some form of built-in professional... ensure your expectations are reasonable, your reagents are onsite before you need them, and you are set... plan as well. Making logistical and funding arrangements Visiting scientist positions take commitment...structured visiting program, you will have to make arrangements on your own. This will include, but is not limited...transportation, living expenses, and necessary reagents. Your primary lab may be willing to cover these...

...of your various blot reagents. There are two types of gradients. Protein gradients use dilutions of your...background. Created with biorender.com   Both gradients can be informative, so spend some time thinking...particular question before running one.  Good on gradients? Excellent! Now, let’s head west, intrepid reader...   Ladders If you need very accurate protein measurements, consider using an unstained protein ladder,...conjugated antibody. If you do not need such precise measurements, you can use a colorimetric ladder, visible ... after your transfer. If you are expecting low amounts of protein, make sure your reversible staining ...wet and semi-dry transfers, you can adjust the amounts of alcohol and SDS in your transfer buffers. To...

...chemicals (biotinylation reagents). There are a variety of biotinylation reagents available that differ ...and thus different reagents are useful for biotinylation in different microenvironments. There are also ...organisms for life. Biotin is only synthesized by plants, most bacteria and some fungi, thus mammals must...unaffected by changes in pH, temperature or organic solvents. Uses of biotin in molecular biology Biotin is...protein purification and immunoprecipitation experiments. Biotinylated proteins or complexes can be easily...immunohistochemistry and antibody labelling. In these experiments, biotin is added to a primary or secondary antibody... ideal for signal amplification during these experiments. During detection, a biotinylated reporter enzyme...

...sequencing large amounts of DNA quickly and accurately. We're sure to see many improvements in a variety ...and sequencing gels, as well as their modern equivalents and see what we can learn from their evolution...DNA identity, size, and abundance. If a scientist wants to know if an organism has a particular gene mutation...confirming the results of cloning or knockout experiments to forensics and clinical diagnostics. One example...individual without sickle cell anemia, two DNA fragments are observed because the DNA has the site where...investigate DNA sequence content by comparing the fragments created by different restriction enzyme digests...allows a researcher to properly orient specific fragments of a DNA sequence of interest to create a physical...

... methods. Across these methods, the following components are needed: γ-Retroviral transfer plasmid encoding...similar to lentiviral packaging methods. The three components described above (envelope, packaging, and transfer...because once in the host cell cytoplasm, the viral components are small enough to enter the nucleus through...slightly different isoforms of these packaging components. Therefore, lentiviruses may not be efficiently...plasmid backbone is meant to be descriptive of the components found within the backbone. For example: pLXSN...particle containing Matrix, Capsid, and Nucleocapsid components. Pol Precursor protein containing Reverse...Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine tract; recognition...