...those ideas and build from those.   Figure 1: Virtual lab meeting? No problem! You can still creatively... on it – so it is important to take breaks. After 1 hour (or another designated time) of meeting, someone... creativity are pivotal in writing publications/grants too. As described by several works (Wuchty et al...and do a quick break to chat. This can be done randomly or manually assigning which participants to group...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...

... There are two methods of neuronal tracing (Fig. 1): one to visualize neurons that send information to...significantly updated in 2023 by Jason Nasse.  References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some viruses (e....e.g., rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but...efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality to...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate an...

...sticks out and can be considered a linker (figure 1). In fact, for several FRET biosensors (yellow cameleon...fluorescent protein are relatively close together (figure 1). Where should the fluorescent protein be inserted...fluorescent protein database." Nature methods (2019): 1. PubMed PMID: 30886412. Mastop, Marieke, et al. "A...et al., 2018). Unbiased, random insertions can be generated with a transposon-based strategy (Sheridan...fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience... This type of interference is known as steric hindrance. For instance, GFP may occlude catalytic sites...sites or motifs that are necessary for post-translational modifications. Despite these potential issues...

...blotting, named for its inventor Edward M. Southern (1), can be used to detect DNA identity, size, and abundance...hours at low cost with restriction mapping (Figure 1). Though some labs may prefer to send DNA directly... serotonergic system and behavior.     References 1. Southern, Edwin Mellor. "Detection of specific sequences...into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization of ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts... control sample. Southern blotting applications range from confirming the results of cloning or knockout...

...refreshed figures made using BioRender.com. Figure 1: PRIME editing Please check out the updated guide... to use CRISPR plasmids in the lab and the vast range of possibilities at their disposal. With this update...CRISPR’s ability to make large genome edits using transposases or the Cascade-Cas3 system. We also describe...

...restriction digest and colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation... doi: 10.1371/journal.pone.0038313. Epub 2012 Jun 1. PMID: 22675541; PMCID: PMC3365890....-mAbs. It was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas...heavy (blue) chains as driven by two CMV promoters (orange). Figure from Andrews, et. al., Elife  A limitation...IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target antigen ...the restriction enzyme BciVI was present in the aberrant light chain sequence, but was predicted to occur...express multiple subclasses of IgG antibody. Broad range secondary antibodies are commonly used in research...

...research like metabolism. Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one...have created molecular biology tools to generate transgenic axolotls for an array of different studies. Elly...popular tools for studying frogs include egg transplants and microinjections. Injections of mRNAs, for...Arabidopsis. There are also important tips for plant transformations and the use of Arabidopsis in the classroom...

...prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who believe...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the...with viruses or transmissible diseases, so we were left with no choice but to quarantine in our houses,...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...we call “asymmetric collaborations.” These are transient partnerships where a researcher makes a small ...blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...

...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ...the non-profit sector are transferrable to industry such as technology transfer, communications, medical... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an...

...gel image includes a 1kb ladder (lane 1) that has bands ranging from about 500bp to 10kb, with the 3.0kb...restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction enzymes which...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...

.... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such ... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...in our cells in the single strand form, but double stranded RNA and RNA:DNA hybrid molecules both exist...arise from a ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly occurring...

...endonuclease activity degrades the displaced strand.   Figure 1: EvolvR consists of an error-prone polymerase...mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M...lineage tracing cells that do not tolerate double-strand DNA breaks by introducing a unique tag into a genomic...

...always guaranteed. To learn more about this process check out our Mouse Modeling blog posts: Part 1: Genetically...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

...blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently induce expression..., they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...small, double stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression (more...LoxP vector.   Turn it off for a little while Transient suppression of expression is faster and easier...expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on forever Permanently...

...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process...Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete ...step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is...

...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and...and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components and...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...need a protease reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed...However, the lab wanted to increase the fluorescence range to better study the spatio-temporal dynamics of ...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...

...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...

... et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system. Image from Xiong...Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing...use of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et...“single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that can...protein by a specific transcription response element (TRE) or kinase translocation reporters (KTRs). Find...interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism, the...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova... engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...

... and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts...An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent protein...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent...fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al (2016) or Thorn (2017). However, the theoretical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...

...modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher... of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for multiplexed...

... to focus, which was extremely effective. Others ran experiments at odd hours, used more secluded workspaces...consistently and accurately run a protocol.   Figure 1: Evolution of a Western blot protocol, from a full...

...non-functional allele. For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides,...10.1038/s41576-018-0059-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA... efficacious to target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...

...are working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...the specific reaction you are running. Transformation Transform a few microliters of your ligation reaction...The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies...fragments If you don’t get any positive clone, try transforming the ligation mix in different bacterial strains...

...method entails arranging all calculated p values from smallest to largest and ranking them (1 being the smallest...error, variance, data ranges. how closely data points fall to each other (see Fig. 1). Alpha values –...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...significance by finding the largest p value where its rank divided by the total number of comparisons performed...– Tukey’s test, Newman-Keuls, Duncan’s multiple range test, and several others. Each test has different...negatives, moderate false positives. Duncan’s multiple range test – Most powerful. Low false negatives, high ...): 126-130 (2012). 10.1097/SMJ.0b013e3182498ad5 Curran-Everett, Douglas. Explorations in statistics: statistical...

...to reach out to potential depositors in two ways: 1. Fill out our "Suggest a Plasmid" form Fill out the...where we can find the plasmid. While we cannot guarantee that everyone we reach out to will end up depositing...

...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...can be shared by publishing it as (part of) figure 1, uploading it as a preprint, or documenting it in ...not by Cranfill et al (2016)). Moreover, good performance in the OSER assay is not a guarantee that using...fluorescent proteins when protein tagging is the goal (Cranfill et al, 2016; Constantini et al, 2012). The localization...

...immunogenicity score (Mattei et.al., 2022).    Figure 1: Generation of immunogenicity scores by predictive...antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z  ...ADAs can inactivate the drug and increase drug clearance, lowering its efficacy and increasing the risk...

...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...

... of two outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal dynamics...library was developed when Carrie was a post-doc in Frank Stegmeier’s lab in Novartis Oncology.  Bhang did...lentiviral vectors with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target...

...none. Best for: targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins...The main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity.... it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering...an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What sets Cas3...locates its target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed...convenient for targeting experiments requiring single strand DNA generation, such as homology directed-repair...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets...

... Hot plasmids covered in this segment Hot Plasmid 1: B. subtilis knock out and knock down libraries. Listen...on the B. subtilis libraries. Hot Plasmid 2: Transcription factor fusions modulate iPSC reprogramming efficiency...

...  Hot plasmids covered in this segment Hot Plasmid 1: Photocleaveable PhoCl protein for controlling molecular...Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen to to the Segmennt of Improved...

...Hot Plasmid 1: The Tol2 Gateway-Compatible Toolbox for quickly generating zebrafish transgenic lines to ...

...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual...

...all of its successes, has many deep-rooted flaws (1). As we discovered through the four workshops at the... Follow David on Twitter @driglar.     References 1. Alberts, Bruce, et al. "Rescuing US biomedical research...Ambika, et al. "Transforming training to reflect the workforce." Science Translational Medicine (2015)...better solution would be to reduce the number of entrants into PhD programs to more closely reflect the ...symposium. Eve Marder, professor of Biology at Brandeis University, has recently published an article ...Among other involvements, he helped to establish grants through the Sloan foundation for postdoc-related...

...2021.   The AID system relies on a transport inhibitor response 1 (TIR1) expressed under a desired promoter...and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes. ...cell surface. When the cells interact, GFP is transferred to the receiver cell, and the receiver cell turns...These libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent.... Check out the new additions.  Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f, now...

...scavenger hunt and the subsequent clue. (Begin with clue 1 below!) You will find five photos this way. At the...Subscribe to the blog to stay in the know!     No pranks, just fun and games this April Fool’s Day on the... to find pictures of Addgene pets with Addgene-branded clothing on the blog. Below each photo, the pet...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...position, I just had a phone interview. I also transitioned between different lab groups without having ... an opportunity to demonstrate technical and transferable skills that make you qualified for the position...experience less stressful. First, you can make arrangements with those you live with to have a quiet place...

...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are especially helpful in...information (more on this below). Plating the transformation on ampicillin plates containing X-gal allows...

...plasmid, based on the assumptions outlined in Table 1.   Of course, this is for the simple insertion of...any restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise...relevant work in your field! However, there’s no guarantee you’ll get a response on the first try (or any...

...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...is the goal of Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender...results, but this result shouldn’t be taken for granted for all research going forward. It’s just not that...

...all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of...is available as a kit from Addgene.   References 1. Chen, Baohui, et al. “Dynamic Imaging of Genomic ...spatial relationship of the two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal...a system has been previously described for transcriptional activation/repression and genome editing. ...

...on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to daughter strands of DNA....modifications). Ready to get started?   Figure 1: Break free from the ball and chain!   Epigenetics...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery...specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started to work...by distinct proteins and result in unclear transcriptional consequences (Sprujit 2013). Enzymatic domains...in the genome and directly interacting with transcription machinery.   Well, I’ve got some good news...

...for the transcriptional activation of endogenous genes and it consists of three components: 1. A nucleolytically...complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...together synergistically, modeled after natural transcription activation processes. Konermann and her colleagues...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman...

...why he chose to go into genome engineering. Chapter 1: Early research on CRISPR and what it's like to work...competitive field. Chapter 2: Wei Leong Chew's swift transition from graduate work to an independent position... Coming up with new project ideas, applying for grants, and mentoring. Chapter 5: Wei Leong Chew's lab...

...the lab running like a well-oiled machine. Lab tip #1: Glove trick Putting on lab gloves can sometimes be...hopefully make your life a little easier. These can range from technical tips as in our episode on Barcoding...

...overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification (Option 2)...Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/978-1-62703-764-8_9. PubMed. More Resources at Addgene:  ...vector + insert may spontaneously religate, this transient construct retains functional Type IIS sites and...