... learned, and ask questions later – maybe even the next day. In the latter case, you can say “I usually...planning you can do to build a bench mentor-mentee relationship and bring your best self to the training experience...plenty of articles on building successful mentor relationships, asking for mentor support, and choosing your...the research group). The bench mentor-mentee relationship is extremely important – without a good set ...mentor than your scientific mentor. Thus, this relationship will probably define your experience in the ...Luckily, you can do a lot to contribute to the relationship dynamic and set yourself up for success! Getting...ll probably forget and end up asking your mentor later if you don’t write it down. And don’t be afraid ...

...chemiluminescence, precipitate the protein out of solution, or isolate cells expressing this protein. Read on to learn...antibodies against the injected protein. Several weeks later, blood from the animal is drawn and the antibodies... hand, monoclonal antibodies are a homogenous population of antibodies that all recognize the same epitope...dilution, which produces an immortalized, clonal population of cells that produce a single variant of antibody...antibodies, recombinant antibodies are a homogeneous population. However, unlike monoclonal antibodies, which.... Western blots are frequently used to compare relative levels of protein expression between cell types... present. The reaction takes place in a 96-well plate, where primary and secondary antibodies are used...

...biologist tool box” for its common use in plasmid manipulation and genetics research. Plasmids are ideal tools...studying genetic modification for their ease of manipulation and their ability to be replicated. Because ...processes naturally found in E. coli have been manipulated for use in molecular biology studies. For example...BSL-1 status, E. coli can be used as a model to relate these processes in more pathogenic bacteria (Blount...rat, is another popular model organism. A close relative to the mouse, both are great mammalian models ...limiting attributes of rats is their difficult to manipulate genome and limited genetic data. To bridge this...biological processes and signaling pathways that regulate regeneration and wound healing are conserved across...

...reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop...control gene expression, he could then selectively manipulate only GFP-labeled cells. Once Tang and Cepko found...but specific system. T-DDOGs also successfully regulated expression of channelrhodopsin-2, commonly used...for optogenetics experiments in specific cell populations. Listen to Our Podcast Interview with Connie ...-DOGs The T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, ...Cepko were eager to see if they could apply GFP regulation to other types of proteins. They started with...system may make optogenetics in specific cell populations even easier. Advantages and other possibilities...

...immunogen, then isolating and purifying the antibodies produced from its serum several weeks later. Created ...as KLH or Freund’s adjuvant is injected, which stimulates the overall immune response. Once the initial... antibody response starts to wane about a month later, the animal is given additional booster immunizations...contains the antibodies - is further processed to isolate the antibodies. Most commonly, antibodies of the...the IgG isotype are used for research purposes. Isolating polyclonal antibodies from the serum There are...are a few different ways the antibodies can be isolated from the serum. Protein A/G purification Protein...different type of purification can be performed to isolate antibodies only recognizing your protein of interest...

... overall inflation of USD has gone up 19.8% in the same time period. For ease of calculations*, let’s ...well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any of the ...Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench’s lab in the...long does it take in total? While much harder to calculate, in both our 2018 and 2023 poll, most people said...much does it cost to make a plasmid? Doench also calculated the cost of consumable reagents used to make ...2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene, the consumables...s split the difference and put scientific inflation at… roughly 34.9%, which means that the same plasmid...

...specific genes involved. Downregulation of tumor suppressors and upregulation of proto-oncogenes ­­­can...while simultaneously stimulating growth and division. The combined effect is unregulated proliferation. Another...cases arise and millions of people die from cancer-related complications each year. Despite these grim statistics...cells still need to successfully infiltrate the circulatory or lymph systems to spread, or metastasize. Many...Many cancerous cells will die trying, as the circulatory and lymph systems are not ideal environments ...Ras researchers, one of the most exciting and relatively new resources is the RAS Pathway Clone Collection...set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) that represents...

...terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host machinery...gene start) and a transcription termination/polyadenylation signal sequence (gene end). A minigenome is...minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP encapsidates the negative...then recognizes the encapsidated minigenome as a template for replication and transcription, as it would... necessary control elements for each would be relatively large. Cloning the viral genes in this plasmid...-minigenome). The anti-minigenome is used as a template by the viral polymerase to synthesize additional...addition, the polymerase will use the minigenome as a template to transcribe the reporter gene (e.g. firefly ...

...location which can all be extrapolated from the 2D barcode and LIMS: Barcoded Tube Plate Freezer Rack Freezer... location within a plate that can hold up to 96 tubes (see image below). Each plate has numbers and letters... Each sample that is transformed is plated onto a petri dish plate that is also barcoded. The barcodes... to give coordinates, from A1 through H12. Each plate is also barcoded with a 10 digit barcode and then...rack that contains five levels that can hold five plates on each level. This rack is located in one of five...sample type. It is critical for us to know which plate, rack, and freezer each sample is located in. Otherwise...identifies the contents of each individual tube.  If a plate of samples is ever dropped, we can simply scan the...

...enzyme specifically cuts methylated DNA, so it will selectively destroy the template, but not newly synthesized...-55 °C for the homologous region (calculated using Oligo Calculator). Insertions - To insert short sequences...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm... bp, such as epitope tags, design primers with template binding regions to amplify away from the insertion...

...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done through... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...is recommended you check your cell lines for HR-related mutations before attempting knock-ins by HDR. ...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...

...not hosted on the platform, they can be hosted on YouTube, Vimeo, and other platforms (such as university...economics, or maybe even biology or international relations.  Many of the challenges in science today are ...appealing to many audiences. While all of these platforms were developing, Google acquired Youtube and has...quickly. Taking what I loved from each of these platforms (such as social networking, visual appeal, instant... across multiple other sites), I created a new platform for sharing video abstracts about research: www.WeShareScience.org...www.WeShareScience.org. WeShareScience is a free platform for sharing short, 3 to 5 minute videos about ...university websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright...

...to the template strand. Therefore, I suggest that picking a crRNA complementary to the template strand...bound by a protein of interest are pulled-down and later coupled to high-throughput sequencing to identify...optimized to achieve high knock-in efficiency. To calculate the tagging efficiency in the transfected cells...tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel tag knock-in for 96 different...single go (Figure 3). We were able to tag >30 genes/plate in the first shot with typical knock-in efficiencies...knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected in 96-well format...device; 5 days after the transfection, the entire plate was scanned for V5 tagging using an Operetta imaging...

...of how miRNAs regulate their targets and what confers specificity in the miRNA regulatory process. This... the then RNAi Platform, which has since evolved into the Genetic Perturbation Platform and encompasses...Associate Director of the Genetic Perturbation Platform at the Broad Institute and has worked with many... up on a Saturday at like 7 am to go check your plates and you’re excited to do so, you know that that...you.” After recognizing his love for science and later realizing that he wanted to continue doing research...The model that the Broad was building that had Platforms with a capital ‘P’ focused on technology ... has...enjoys working on technology development under the Platform model at the Broad Institute because they are ...

...microscopy methods will be used to stimulate and manipulate neural populations in order to get new insights ...current research, Sheen aims to identify new genes related to PZA-resistance by using different approaches...understanding of PZA action and could potentially stimulate the development of useful diagnostic methods and...collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract infections. This project...to create gene deletions of important unknown regulatory regions in Pseudomonas and Klebsiella spp. The... several diseases. Her goal is to identify and isolate pure plant compounds with antidiabetic properties...Recombinant human proteins that play a role in insulin regulation and diabetes will be expressed from plasmids ...

...for post-translational modification of recombinant protein products, specifically glycosylation. The native...eukaryotic model organism – its genome is easy to manipulate, it’s affordable, and it grows fast to boot. ...doubling time of ~ 2 hours) Amenable to genetic manipulation The first eukaryote (S. cerevisiae) to have...glucose or glycerol). K. phaffii is only distantly related to S. pombe and S. cerevisiae; evolutionarily it...native glycosylation machinery of K. phaffii lacks certain terminal mannose glycopeptides (which most ...in K. phaffii since it produces ‘human-like’ glycosylated proteins. Peroxisome studies Peroxisomes are...biogenesis, degradation, and proliferation. Genome manipulation K. phaffii’s genome has been sequenced for some...

...investigate the effect of regulatory elements, such as promoters, enhancers and untranslated regions, or the effect...effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type or mutant...Higher Lower Background Lower Higher Post-translational Modification Not Required Required Photobleaching...Biological sources of luciferase Although luciferase isolated from the firefly beetle (Photinus pyralis) is ...Luciferin ATP No Japanese firefly(Luciola cruciate/lateralis)Italian firefly(Luciola italica)Southern Russian... produce light, often with a luminometer or scintillation counter, and the activity measurement can then...then be used to quantify the effect of the regulatory element. Depending on the specific luciferase used...

...the recombinase handles the installation of the donor sequence. The latest work evolves and engineers ...D3L, releasing the autoinhibition and stimulating the methylation of nearby CpG sites on the target DNA...problematic for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki...Funabiki Lab developed a technique called Magnetic Isolation and Concentration (MagIC)-cryo-EM that combines...streptavidin-coated magnetic beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins...in sample loss. Ci Ji Lim’s lab found that adding Late Embryogenesis Abundant (LEA) proteins, small proteins...particle orientation distribution and Fourier-shell correlation curve (D), and the reconstructed 3D map with ...

...detected using lateral flow strips. Since the probes are tagged with streptavidin, the lateral flow strip ...concept to lateral flow strips that contain streptavidin which can bind to the biotinylated targets. They...earlier epicenters of the outbreak ultimately flattening the curve, it has become evident that widespread...fluorescence and this approach can be incorporated into a lateral-flow readout slip that allows rapid detection.  ...substrate, gets activated and performs non-specific collateral cleavage. This cleavage is coupled to fluorescence.... In this technique, however, instead of using lateral flow strips to detect the cleavage of fluorescently...hours Equipment needed: PCR machines/Heat blocks, Lateral flow strips, Agarose gel equipment, Fluorescent...

...receptors, or post-translational modifications on those proteins, like glycosylation patterns. Surface ...only be interested in one of the various cell populations; for example, CD8+ T (“T killer”) cells. Typically... simply “dump” them by gating on the negative population.     Figure 2: Dump gating labels all...allowing for a flow analysis to be shifted to a later day. Note that intracellular labeling must be performed...method, differences in proliferation between cell populations can be visualized, achieved through, for example...heterogenicity of proliferative behavior in a cell population can be seen as a stepwise decrease in signal ...easily gated on during the analysis. Conclusion Congratulations! You made it through this blog post and hopefully...