...long-lasting way to identify and reference documents, files, or physical reagents. What are persistent identifiers... there is insufficient information about which reagents were used? Unambiguous identification of a reagent...Object Identifier (DOI). Nearly all the papers or preprints you read or produce have one. The DOI serves as...publications help to enable reproducibility of experiments. Additionally, proper citation of these Addgene...standardized persistent identification of research reagents, several journals or organizations now recommend... the RRID and compact identifier formats. Acknowledgments Many thanks to Daniela Bourges for editing ...

...that’s where MTAs, and similar agreements, shine.  These types of agreements allow for centralized tracking...and developments.  MTAs also mean that everyone is on the same page as to how the shared reagents should...organizations to make policy decisions on research use agreements. The UBMTA, which is the MTA most plasmids at...department is responsible for managing research use agreements like MTAs.  Addgene does a lot to ensure that...tracking when biological reagents are shared — both at what time and with whom. Tracking these connections...

...in silico. These algorithms are trained on vast amounts of data to calculate the relative immunogenicity... replicate the complexity of antigen processing events that take place within an antigen-presenting cell...conditions, etc. that can vary widely between recipients.   In vitro prediction of drug immunogenicity... to validate the findings from the in silico assessments, as well as analyze the contributing factors ...in-vitro models can be built that contain various components of the immune system. Figure 2: In-vitro...Potential Use of HLA Haplotypes to Predict Vulnerable Patients. Frontiers in Immunology, 13. https://www.frontiersin.org...Algorithm Infrastructure and a Vision for Future Enhancements. Frontiers in drug discovery, 2, 952326. https...

...or the use of alternative substrate reagents can permit measurements of luminescence from live cells. Monitor...various species with differing kinetics, substrate requirements and photon emission wavelengths. Renilla luciferase...often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...or the effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type...measure of signaling activity. Multiple response elements are cloned into a plasmid upstream of a minimal...in luciferase expression and more complex cell treatments can be designed to further explore the pathway...

...cells. Before getting into the mammalian plasmid components, it may be useful to describe the means of introducing... bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells...transfection. Unlike bacterial transformation, scientsts do not "select" for cells that have taken up the...transfection and the elusive "mammalian ORI" For many experiments, it is sufficient for the transfected plasmid...plasmid replicates in the cell, as many of these experiments are concluded within 48 h of transfection. Of...void. These ORIs, however, require additional components expressed in trans within the cell for effective...ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution ...

...each level, as each level has different safety requirements. BSL-1 is designated for those working with ...

...competitive endeavor. Due to the COVID-19 pandemic, students will face a reduced supply of internships as a... to the opportunities of their dream.  To help students focus on the application process, and simplify...internships for high school, college, and graduate students. In order to gain a competitive advantage this...errors, sentence structures, and overall format (fonts, font size, legibility, and more). Tips on drafting...leadership status in their current field of expertise augments their authoritative voice. Send a draft of your...in the Chemical Biology Program at Harvard. Roo wants to simplify access to academic resources among economically...outreach among low-income communities to educate parents about resources that can help their children thrive...

...their required components are very similar. While plasmids containing virus-specific elements such as the...or infrequently cycling cells but is fine for experiments with immortalized cell line cultures. Lentivirus...between the two systems, other, more general, viral components such as a heterologous envelope or a post-transcriptional...whole animal administration, such as in mouse experiments. AAV also has multiple serotypes (12 naturally...strategies may need to be considered if size constraints are an issue. Adenovirus Adenovirus has a linear...Addgene blog Viral Vectors 101: Viral Vector Elements Viral Vectors 101: An Introduction to AAV Viral...

... but should contain a table of contents that lays out which experiments can be found in each folder. This...their results. We are adept at recording wet-lab experiments using a “traditional” lab notebook; however, ...the future. Stay organized Set up a table of contents in the top directory: A directory is a file system...document with a brief description of the directory contents, at a minimum. This may or may not be where you...in tact! Take notes Comment in your scripts: Comments on what a given line of code does are very useful...Software You Can Use to Save Time and Track Your Experiments Try out the CRISPR Software Matchmaker Additional...

...Before you jump straight into the exciting new experiments that you’ve been planning, it is a good idea ...yourself have to perform extensive validation experiments for every antibody that you receive; citing existing...However, it is your responsibility to determine what counts as adequate validation. If you are not convinced...your sleeves and perform some extra validation experiments yourself.  Goal of Antibody Validation  As you...assess existing data or plan your own validation experiments, it’s important to know what you are aiming to...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental... that your new antibodies will work for your experiments. Capture MS Strategies This approach uses immunoprecipitation...

...professor who once decided that to teach high school students PCR, they first had to understand hydrogen bonds...every project, time, attention, and/or length restraints mean you will have to pick and choose what concepts...when needed. The trick is to communicate your key points well enough that the audience can take that knowledge...Different modes of communication take up different amounts of mental bandwidth - if not exactly cognitive ... audience, ask! They are experts in what their students know.   Ask the learners themselves for feedback... about the questions you’ve received, read the comments, seek feedback from others, and listen quietly...simpler presentation than I had planned; other times students were fired up and enthusiastic and needed a faster...

...deliver these components together using electroporation. In these bulk cell experiments, Clift et al. ...proteins, and it can even distinguish between protein variants when used with a variant-specific antibody. TRIM21...need to deliver the target antibody. TRIM-Away reagents and reporters are available from Addgene - we ...down the difficulty of your protein knockdown experiments! The TRIM-Away plasmids can be found here. Clift...varying which sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once...factor. They then induced cultures of these transformants with various concentrations of estradiol and...

...Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole...double-membraned structures containing undigested cytoplasmic contents. Autophagosomes that have fused with the vacuole...lysosome are trickier to identify because their contents can be at various stages of degradation. Additionally...Atg8p or LC3 doesn’t provide information about the contents of the autophagosome nor does it provide information...sequence. Under normal growth conditions, both variants are excluded from the vacuole and emit red and...the cytosol-targeted and mitochondria-targeted variants are available from Addgene. References 1. Rosado... L. K., Graham, T. E. (2015). Rapid parallel measurements of macroautophagy and mitophagy in mammalian...

...line./ Each color represents a gRNA targeting a specific chromosome. This image represents the composite ...composite of fluorescent measurements from red, blue, and green channels overlaid on a bright-field image. Image...fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency was similar to that of ... other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences and...distance! To the authors’ knowledge, this work represents the first mapping of intrachromosomal loci, a... live cells. However, they note that certain improvements could make CRISPRainbow even better. Adding ...

...specific components that are key to the cloning reaction. Understanding the specific components is essential...choosing the correct cloning method for your own experiments, and here we will focus on a unique gene that...on to find out how ccdB can make your cloning experiments a little easier. One of the most time-consuming...choose the most appropriate method for your own experiments. Please check out Addgene’s Choosing a Molecular...these resources further. Feel free to offer your comments and suggestions, and even to submit a guest blog... al. “F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain ...

...washout experiments (Nabet et al. and Weintraub et al.), chemical and genetic rescue experiments (Nabet... for degrading targets in different cellular compartments, and in vivo utility. dTAG molecules will be... line of interest prior to starting knock-in experiments. Test dTAG molecule toxicity in new cell lines...is recommended, while quantitative proteomics experiments will provide the best confirmation of selective...-substrate degradation with dTAG-13. Example experiments See our recent papers noted below for examples...Nabet et al., Erb et al., Weintraub et al.), assessments of fusion chimera functionality (Nabet et al....

...dimerizes bringing the C-terminal and N-terminal Cre fragments together and permitting recombinase activity. ...Biggiephage can eliminate competing mobile genetic elements. Image by Basem Al-Shayeb in the Doudna Lab. ... more compact genome editors due to delivery constraints and scientists seeking a wider range of targetable... to image over 3 million cell types expressing variants of Yellow Fluorescent Protein (YFP). The team ... As such, mGold is expected to contribute to experiments that require high photostability, such as time-lapse...compared to current N-terminal linked SaCas9 ABE variants. It's also one of the smallest AAV-deliverable...dLight1.3b here Looking for a knock-out in your experiments? AAV-flex-taCasp3-TEVp from Nirao Shah's and ...

...correction in adult stem cells isolated from two patients with cystic fibrosis (Schwank et al). They were...giving this treatment a CRISPR spin. CRISPR CAR-T treatments for leukemia, lymphoma, and some solid tumors...these trials has been outstanding, with almost all patients experiencing sustained and substantial relief ...are being finalized, and it is very likely the treatments will be approved for use in the next few months... a desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One treatment...just don’t have the time to talk about. Other treatments currently in clinical trials include, but are...Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (2013). Dimitri,...

...to split it into multiple fragments. You can define the number of fragments, and the “search windows” ...number of fragments, you can also ask it to give the maximum or minimum number of fragments, or defined...assembled a 40 kb T7 phage genome from 52 (!!) fragments in one very Golden (Gate) pot (Pryor et al., 2022...high-fidelity assembly for sets of up to 10-12 fragments (Pryor et al., 2020), and randomly selected non-palindromic...reaction time of 15 hours. This suggests that 52 fragments may be near the upper limit of what can be accomplished...

...One such control is dividing up the essential components of a virus into different plasmids. For a retroviral...enter (and thus infect) cells, this safety feature prevents the virus from replicating and spreading in non-target... Figure 1: Separation of retroviral packaging components onto distinct plasmids for safety.    Viral...biohazard policies. Administering Virus Many virus experiments involve needles or scalpels (sharps). Cuts and...equipment are one of the most common forms of lab accidents, so extra precaution should be used to reduce ...of an accident (the most common reason for lab accidents is not following safety protocols.) If administering...Viral Vectors 101: Types of Viruses Viral Vector Elements Tips for Preventing Viral Recombination  ...