...backbones for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning vector. Lentiviral Plasmids ...they were added to the repository, including beta-catenin reporter M50 Super 8x TOPFlash, scramble shRNA ... GCaMP6 series of genetically encoded calcium indicators were the hot technology of the year in our collection...

...nucleotides are represented equally, that’s only a 1/16 chance of having a PAM at a given site. While it... NYN/NRN NYN is less effective than NRN Table 1 - Summary of PAM-flexible Cas enzymes Are we truly...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome...flexibility in the location of the cut and subsequent edit. In these applications, location takes priority...make a choice about what is your top priority: location or efficiency. If you are just trying to shred...still leave you choosing between efficiency and location.   Nearly PAM-less editing   The Kleinstiver lab...in double stranded cleavage and base editing applications, with high editing capacity in both contexts...

...targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...active in retinal cell types in mice. Figure 1: The four strategies used to design synthetic promoters...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry...

..., check out Addgene's CRISPR Guide. Figure 1: Like this jar of jellybeans, a pooled CRISPR library... selecting a CRISPR library for your experiments. 1.  What species are your cells derived from? Currently...a different gene. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-...

...unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively new technology that takes...conveniently modified and engineered.   Fig. 1: Antibody production methods    NeuroMab and others... the signal. If the exposure time is long (e.g. > 1/7s) then the signal is weak and vice versa for a strong...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...

...-public settings. Ask over coffee.  Have regular 1:1 meetings with team members (don’t become known for...Scientists series will focus on an aspect of communication especially important for a manager – getting... Creating a culture of excellent, effective communication between all members of a team. As described ... opinions in meetings, via email, at formal communications events or at informal gatherings, like at lunch...

...about HIV cure research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. ...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID: 25665714...our AAV Production Protocol Check out our AAV Purification Protocol ...

...depend on your intended application and available resources.     Figure 1: Table summarizing the... a good option for applications like immunohistochemistry on human tissues, when (1) other strategies ...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...validation: A view from the mountains. New Biotechnol 45:1–8. https://doi.org/10.1016/j.nbt.2018.08.002  Uhlen...intended application - Remember, just because your antibody performs as expected in one application does ...strategies for antibody validation in different applications. Here, I want to highlight the general suggestions...intentionally generalizable and not specific to certain applications or antibody targets. That said, not every strategy...

...School Advice Part 1: Picking a Lab and a Project That’s it for now. As I indicated in the opening paragraph... products). I picked this post for a few reasons: 1) I think phage are super cool and have considered ...Plasmids at a Low Price: Kits from Addgene Savvy Advocates Needed to Navigate a Scientific Enterprise in ...

...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central... also been developed which don’t fit in either category, but those are a subject for a future post! ... GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further... PMCID: PMC2942869. Find plasmids from this publication at Addgene. del Valle Rodriguez, Alberto, Dominic...

...initiating cleavage of the transcript.   Figure 1: A predicted conserved secondary structure and sequence...prokaryotic terminators and deposited several with Addgene (1). Prokaryotic polyadenylation Although mostly thought...constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic...type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional...termination mechanisms fall under two general categories: rho-dependent and rho-independent. Rho factor...

...” Nat Protoc. 11(1): 118-33. PMID: 26678082. Find the plasmids from this publication at Addgene. 2. ...homologies. Abbreviated protocol for PITCh Step 1: Generate microhomologies in the PITCh vector ~20...editing possibilities MMEJ can enable. References 1. Sakuma, Tatsushi, et al. (2016).  “MMEJ-assisted gene...another method for making targeted modifications. A recent publication by Zhang et al. shows just that -...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins... and 3’ junctions, respectively. These results indicate that PITCh is a robust method for genomic insertion...

...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 ...vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 ...

...some technical obstacles when working with virus: 1. Viral vectors undergo DNA rearrangements. Depending...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...up-to-date tips for your virus experiments. References 1. Anthony, Simon J., et al. "A strategy to estimate...experiment and they don't do anything. Viruses are complicated and they perform complex biological tasks, both...vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying...membranes (Davis et al., 2002). Polybrene is one such cationic reagent that is commonly used and has been shown... during transfection, and does not necessarily indicate the production of virus. Alternatively, if the...

...cell with the images from the Allen Cell Explorer (1). When studying the function of a protein or its role...various subcellular structures. Please see Figure 1 for commonly used markers for major mammalian organelles...Subcellular Localization Poster here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000....functionality can be gained by understanding the location and transport of the protein within a living cell...to observe membrane trafficking events in vivo. Locate your protein and see who it is hanging out with...potential overlap, it is possible to determine the location of proteins within complex structures. In order...way it is important to choose the appropriate quantification methods and tools. It is usually not enough...

...active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components. Image from Peters...on a non-selective plate. Most bacteria tested had 1 transfer in every ~100 to 1,000 colonies, but some...including E. faecalis and P. mirabilis, had only ~1 transfer per 1,000,000 colonies. Higher transfer rates...The vast majority of bacteria are undomesticated which limits the tools scientists can use to study them...the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt...single-gene knockdown experiments. Mobile-CRISPRi applications Mobile-CRISPRi expands CRISPRi to non-model ...Elizabeth A., and Pamela A. Silver. "Harnessing undomesticated life." Nature microbiology 4.2 (2019): 212....

.... pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors ...your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...

... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...to rapidly punch out cell clones with sterile truncated pipet tips. Indeed, an experienced operator can...wiped down with 70% ethanol, and a box of sterile truncated pipet tips (USA Scientific #1011-8410) is also..., and making sure we always maintain a backup replicates of each clone in case of contamination. We do...

...biological questions. The two most common uses are: 1) to test the expression level in a specific system...brightness is compared to that of EGFP which is set as 1. Some proteins are very dim (e.g. TagRFP657, which...Photostability can be as short as 100ms (EBFP) or as long as 1 hour (mAmetrine1.2). However, for most FPs it is a... to be considered when choosing an FP: Unique categories of fluorescent proteins Photoactivatable / Photoconvertible... used are GECIs - genetically encoded calcium indicators (e.g. GCaMP). Others include: pHluorin & pHTomato.... al. (2010) “Fluorescent proteins and their applications in imaging living cells and tissues”  Physiol...

...optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference... plasmids are available from Addgene! References 1 Bourdeau, R.W., Lee-Gosselin, A., Lakshmanan, A., ...Knowing where bacteria are located within their host is often key to understanding their role in both...reporters are not as well suited for imaging organs located deep in the body, like the gastrointestinal tract...lab recently discovered that gas vesicles also scatter sound waves and therefore produce signals in ultrasound... experiment to demonstrate the most exciting application for ARG’s: in vivo imaging. Specifically, arg1...