...two weeks, we were able to obtain eight clones. We ran digested and undigested samples of the clones on ...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...and I wouldn’t want it any other way. Toj Toj-Villagran is a rising senior in high school who just completed...

...nLightR (Kagiampaki et al., 2023).     Figure 1: Properties of nLightG and nLightR. A, Structural ...Article 6. https://doi.org/10.1038/s41592-022-01488-1 More resources on the Addgene blog Fluorescent proteins...generating variants with improved affinity and dynamic range of both nLightG and nLightR, and may soon have a...

...following three strategies might help!     Figure 1: Species on species staining can be a problem because...same host species as your secondary antibody since 1) your secondary should not recognize Fab fragments...References and Resources References Goodpaster T, Randolph-Habecker J (2013). A Flexible Mouse-On-Mouse Immunohistochemical...

...your budget, you should take the following steps: 1. Define your timeline Start with your thesis in mind...these decisions in the following order of importance: 1. First make decisions based on experimental timelines... several factors aren’t taken into consideration. 1. Always focus on the science When negotiating finances...will take to secure your first (R-level) research grant.  Set your timeline from there, then start identifying...techs’ financial books.  To find personnel salary ranges, go to online job information sites (like, indeed.com...

...the journal-specific guidelines.  Peer-review step 1: The first read At the first read of the paper, I ...group of Prof. Mark Tester, where she focuses on (1) salt-induced changes in root-to-shoot ratio in Arabidopsis... discipline and reduces the dissemination of unwarranted claims or unacceptable interpretations. A good...studying plant development and abiotic stress tolerance, I have been peer-reviewing journal submissions...

... by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...

...sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson et...):1028-34. doi: 10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed. Find plasmids from this publication at ...can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just...markers. Bob Goldstein’s lab previously used a transgene selection strategy for their C. elegans/CRISPR...

...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...