... hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols...deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...remove one strand of the siRNA and are directed by complementarity to the remaining single stranded siRNA ...activity or their translation disrupted through physical blocking of the translation machinery. In the...WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to...

...C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1, but ...lacks the ability to cleave the RNA target.  Table 1: Comparison on Common CRISPR Enzymes Name  Enzymatic...Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of DNA ... This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus...contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...Recombinase Polymerase Amplification (RPA) or Reverse-transcription RPA (RT-RPA), respectively. Amplified nucleotides...

...and plant transformation. Hortic Res 7, 152 (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... be collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...

... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert a ... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the... mechanism to accurately repair harmful double stranded breaks, in which nucleotide sequences are exchanged...selection marker HSV-tk is used to select against random recombinants.   Design your targeting construct...selecting for cells in which the construct has randomly integrated into the genome, a negative selection...step will kill off many of the cells that have randomly integrated all or large portions of the plasmid...

...events by HDR can be generated more efficiently by (1) improving editing accuracy by preventing re-editing...specifically incorporate homo- or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking ...targeted alleles when gene editing.   Figure 1: Techniques to improve editing efficiency.   But...provided by blocking mutations. Essentially, you:  Step 1 - Introduce the blocking mutation together with the...developing novel iPSC-based model systems.     References 1. Cong, Le, et al. "Multiplex genome engineering using...com­plex is very efficient at introducing double stranded breaks (DSBs) into genomic DNA in many cell types...homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the...

...milliliter can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas...nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks...the zeptomolar range while One-step-SHERLOCK has a sensitivity in the femto-aptomolar range. For more details...are designed, each binding to one strand of the reverse-transcribed target sequence that are present close...

...photosensitizer used in this work is composed of two parts: 1) an activatable protein (the FAP - fluorogen activating...reaching greater than 75% photobleaching after just 1 minute of exposure. MG-2I-dL5** experienced minor ...photosensitizers in the comments below!   References 1. He, J., Wang, Y., Missinato, M. A., Onuoha, E., Perkins... encoded photosensitizer. Nature Biotechnology,24(1), 95-99. doi:10.1038/nbt1175. PubMed PMID: 16369538...these experiments because developing embryos are transparent, so it’s easy to watch development in real time...

...Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but with mOrange2, EGFP...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...subsequent recombinations cannot occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of...antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...

...intended.)  Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words...information as sighted readers.    Figure 1: An image from AAV Purification by Iodixanol Gradient...are they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification ... share with the rest of the workshop.  For Figure 1, Addgene chose this alt text: "The left panel is a...resources on the Addgene blog Five Steps to Writing Grant Proposals to Grad Students Writing Scientific Manuscripts...

...how scientists connect on Twitter that I realized 1) a lot of scientists are using social media professionally... but my consistent writing for the Addgene blog (~1-2 blog posts per month) and use of Twitter and LinkedIn... have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast...of interest. Hopefully through these links, I’ve: 1) convinced you social media can positively impact ... to research potential future employers and to arrange informational interviews. 4. “Tips for Scientists...

... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based...

...perform, at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation...competent cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent ...well, so let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies,...result in few colonies on both plates (unsuccessful 1), in a vector alone plate with many more colonies ...contamination from uncut plasmid. 4. Transformation Transform your ligation reaction into your bacterial... how many colonies you should expect in the transformation due to background re-circularization and contamination...

.... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples...backbone might be more difficult to transform.    Figure 1: Factors to consider when creating a...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...

...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

...2021).   Table 1: Properties of modern NIR FPs     NIR FP Ex, nm Em, nm ECa, M-1cm-1 QYb, %   Molecular...expression level. State-of-the-art FPs (listed in Table 1) were optimized for efficient binding of endogenous...previously reported widely used dimeric iRFPs (Table 1). In general, the monomeric state of a probe leads...iRFPs in cellular and molecular brightness (Table 1).  NIR reporters and biosensors NIR reporters can ...dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with no supply of exogenous...lamps are the best for working in this spectral range. You can also check that no filters and/or cold ...and after normalization to fluorescence of co-transfected EGFP. e Originally reported as a monomer16, IFP2.0...

...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater number of neurons ...intravenous gene transfer throughout the non-human primate brain. Nature Nanotechnology, 1–11. https://doi.org...endothelial cell transduction throughout the central nervous system. Nature Cardiovascular Research, 1(4), Article...capsids facilitates enhanced transgene expression in the macaque CNS. Med, 4(1), 31-50.e8. https://doi.org...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...

...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...chromoproteins are just now being developed for a broad range of biological inquiries.  What is a chromoprotein... a gene encoding a blue chromoprotein, after transforming them into your cells, you can simply look for... plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue...Image: Murakoshi et al., 2019. ShadowR is an orange light-absorbing chromoprotein that Hideji Murakoshi...

... different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail...follow him on Twitter @derekpsimonphd.    References 1. Gutierrez DV, et al. Optogenetic control of motor...manipulation of neural circuits. Nature protocols. 2012;7(1):12-23. doi: 10.1038/nprot.2011.413. PubMed PMID: ...freely moving mice. Neuroscience research. 2011;70(1):124-7. doi: 10.1016/j.neures.2011.01.007. PubMed ...tissue, placement above the site of stimulation guarantees that the majority of the opsins will receive ...tangled. Furthermore, to give the animal the full range of motion, a commutator allows the patch cable to...

...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...pattern opens up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning... cloning. Sticky-end mediated gene transfer would be particularly helpful for targeting non-dividing cells...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...

...genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula J....which are required for replication and transcription (Figure 1). All viral genes are removed and replaced...negative strand RNA viruses: The power of reverse genetics. Springer Berlin Heidelberg, 2004. 1-41. PubMed...cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes will greatly enhance our...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...