...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0...Biosystems offer pre-made stripping solutions or kits. Mild stripping The mild stripping solution uses low pH...lose by trying to strip and reprobe. In this case, we recommend using mild stripping solution (see below...later, strip it thoroughly first. Then continue on with your western blot as normal! Stripping your membrane... to strip blots, and the best choice will depend on your specific experiment. Remember, stripping your...buffer such as PBS or TBST. Stringent stripping The stringent stripping solution uses heat and detergent to... start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you can remove...

...currently on my way back from a very successful Outreach Trip in San Francisco and would like to give my thanks...who spoke with me and deposited plasmids on this trip. Here’s to hoping that October is even more awesome... products). I picked this post for a few reasons: 1) I think phage are super cool and have considered ...Connect the Research Community Grad School Advice Part 1: Picking a Lab and a Project That’s it for now. As...

... dark matter and so the lab organized a sampling trip to Yellowstone National Park (YNP). Extremophiles... another Quake Lab member joined for half of the trip. Preparations ahead of time included contacting ...could be kept frozen after collection. Figure 1: Paul included a voltmeter on top of the battery box...

...you might need different plasmids. Harvard’s DRSC/TRiP Functional Genomics Resource Center has various ... you’d like to use to generate your new fly line: 1. Where would you like the gene to be incorporated ...control exactly in which locus your transgene ends up (1): Random insertion has the advantage that you can...he still reads “#” as a “pound symbol”. References 1. J. R. Bateman, et al. Site-Specific Transformation...

...many genomic locations. In our study, we used the TRIP (Thousands of Reporters Integrated in Parallel; ...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...

...start working with live animals yet, Harvard’s DRSC/TRiP- Functional Genomics Resources website has in vitro...

...that I started writing three years ago on a field trip in Indonesia. Since then, I have tried many things...started:  Subscribe to Career Blog Posts from Addgene 1) Twitter – Get your voice out there Here’s one thing...

... every school performance, chaperone every field trip or cheer at every soccer game. Tip: take turns going...commandments for making it all work – most of the time. #1 Thou shalt find enjoyment in your work If you are ...

...190293 Anti-AMIGO-1 [L86/14] AMIGO-1 Mouse Mouse IgG2a 190294 Anti-AMIGO-1 [L86/2] AMIGO-1 Mouse Mouse IgG2a...ATPase 1 [L60/4R-2b] Copper ATPase 1 Human Mouse IgG2b 206648 Anti-AMIGO-1 [L86/33R-2b] AMIGO-1 Mouse ...channel [L6/60R-1] Slo1 K+ channel Mouse Mouse IgG1 206695 Anti-AMIGO-1 [L86/33R-1] AMIGO-1 Mouse Mouse IgG2a...Anti-Olig1 [N149/25R-1] Olig1 Mouse Mouse IgG1 206702 Anti-Neurexin-1-Beta [N170A/1R-1] Neurexin-1-Beta Human Mouse...222160 Beclin-1 [N248A/15R] Beclin-1 Mouse Mouse IgG2a 222161 Beclin-1 [N248A/22R] Beclin-1 Mouse Mouse ...Mouse Mouse 190534 Neurexin-1-Beta scFv [N170A/1] N170A/1 scFv Neurexin-1-Beta Human Mouse 190535 PSD-...Mouse 206755 IP3 receptor, type 1 scFv [L24/1] L24/1 scFv IP3 receptor, type 1 Rat Mouse 206756 VSP scFv [...

...IGHDY1 IGHD1-1 immunoglobulin heavy diversity 1-1 IGHD11 IGHD1-14 immunoglobulin heavy diversity 1-14 (non-...IGHV6-1 immunoglobulin heavy variable 6-1 IGHV61, VH IGHV7-4-1 immunoglobulin heavy variable 7-4-1 IGHV7... TAPBP-R, TAPBPR THBS1 thrombospondin 1 THBS, THBS-1, TSP, TSP-1, TSP1 TRPC4AP transient receptor potential...variable 2-8 IGLV28, V1-2 IGLV3-1 immunoglobulin lambda variable 3-1 IGLV31, V2-1 IGLV3-10 immunoglobulin lambda...NCC-1, NCC1, SCYA13, SCYL1 CCL14 chemokine (C-C motif) ligand 14 CC-1, CC-3, CKb1, FLJ16015, HCC-1, HCC... 3 G0S19-1, LD78ALPHA, MIP-1-alpha, MIP1A, SCYA3 CCL3L1 chemokine (C-C motif) ligand 3-like 1 464.2, D17S1718...GPD, GpFy, WBCQ1 DEFA1 defensin, alpha 1 DEF1, DEFA2, HNP-1, HP-1, MGC138393, MRS DEFA3 defensin, alpha...

...accumulation in bacteria. Biotechnol Biofuels. 2008 Jun 3. 1(1):11. Wolf Frommer Maltose Green fluorescent MBP-based...of cellular physiology. Nat Commun. 2020 Aug 4;11(1):3881. Adam Cohen Calcium GCaMP6f expression in forebrain...Dynamics in High-Ca Organelles. Cell Chem Biol. 2016 Jun 1. pii: S2451-9456(16)30163-5. Teresa Alonso , Javier... intracellular calcium. Nat Commun. 2021 Dec 9;12(1):7159. Dorus Gadella Calcium Teal genetically encoded...Neurophotonics. 2024 Apr;11(2):024207. doi: 10.1117/1.NPh.11.2.024207. Robert Campbell Calcium Ratiometric...orange-emitting fluorescent proteins. Nat Commun. 2017 Sep 5;8(1):431. Wolf Frommer Calcium Red-, yellow-, or cyan-...dynamics in mitochondria. Sci Rep. 2020 Feb 18;10(1):2835. Masamitsu Iino Calcium Red fluorescent calcium...

...Brown 11908 pEGFP-N1 alpha-actinin 1 Actin Filaments alpha-actinin 1 EGFP Carol Otey 27676 Cortactin-pmCherryC1...Huttenlocher 11908 pEGFP-N1 alpha-actinin 1 Focal Adhesions alpha-actinin 1 EGFP Carol Otey 31923 pPaxillin-PSmOrange...Autophagosome Sequestosome-1 EGFP Noboru Mizushima 38272 pMXs-IP GFP-WIPI-1 Autophagosome WIPI1 EGFP Noboru...apparatus eNOS(1-33) CFP Alexandra Newton 36205 pmTurquoise2-Golgi Golgi apparatus B4GALT1(1-61) mTurquoise2... Shirihai 158003 pCMV-mGold-CAF1-C-10 Nucleus CAF-1 mGold Francois St-Pierre 27705 CAV1-mCherry Caveolae...COXIV-COX8-dL5-2XG4S-mCer3 Mitochondria COX IV-derived (1-22 aa) import sequence and COX VIII signal peptide...Verkhusha 36208 pmTurquoise2-Mito Mitochondria COX8A(1-29) mTurquoise2 Dorus Gadella 26673 pEGFP-C1-Fibrillarin...

... with CRISPR activators. Nat Commun. 2018 Jul 6;9(1):2643. Otonkoski Lentivirus Human Expression of human...cell reprogramming. Stem Cell Res Ther. 2017 Jun 5;8(1):132. Zovein Replicating EBNA1 episome Human Non-integrating...vector. Proc Natl Acad Sci U S A. 2009 Jan 6. 106(1):157-62. Jaenisch Lentivirus Mouse Doxycycline-inducible...transcription factors. Stem Cell Reports. 2015 Jan 13;4(1):25-36. Broccoli Fibroblasts Sensory Neurons Lentiviral...peripheral sensory neurons. Nat Neurosci. 2015 Jan;18(1):25-35. Baldwin Fibroblasts iTSCs Lentiviral Mouse... from Human ALS Patients. Cell Rep. 2016 Jan 5;14(1):115-28. Zhang Adult Dermal Fibroblasts Neurons Lentiviral... into Neurons. Curr Protoc Cell Biol. 2018 Jun;79(1):e51. Next generation vectors for transcription factor...

...populations defined by multiple parameters. Figure 1: Examples of intersectional cell population definitions... as a proof-of-concept targeting approach in 2014 1 (using EYFP and ChR2-EYFP as payloads). This approach...interneurons encode fear memory. Nat Neurosci. 23(1):61-74. PubMed (Link opens in a new window) Hafner...Itch-Scratching Cycle via Descending Regulation. Neuron 101(1):45-59. PubMed (Link opens in a new window) Lazaridis...TVA-mCherry Cre AND Flp pAAV-nEF-Con/Fon OG Cre AND Flp Triple recombinase-dependent Addgene ID Plasmid Logic ...

... Figure 1: (A) Representation of a zinc finger (ZF) nuclease. Zinc fingers recognize triplets and the ...zinc fingers that recognize various nucleotide triplets. With some trial and error, ZF arrays are able...their limitations, however. Not every nucleotide triplet has a corresponding zinc finger, and interactions... targets are confined to sequences composed of triplets with corresponding zinc fingers, potential targetable...

...from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from 1:1,000–1:100,000...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). You’ll want to...recommended dilution range is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...Here, the lanes are lightly marked on the membrane (1) using the gel as a guide. The gel is then removed...membrane is reassembled and imaged. In this example, a 1:1,000 dilution gives a high signal with low background...

...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...gel chemistry and/or specialty gel options. Table 1: Types of gels  Gel Type Protein sizes Running... Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...

...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...dilution series should be run in duplicate or triplicate. The closeness of the data points generated by... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve...

...mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks, Lateral flow strips, Agarose gel equipment... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas... can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and 42°C ...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...lateral flow strips. Since the probes are tagged with streptavidin, the lateral flow strip first captures...be captured by an antibody further along on the strip. Highlights: Of the 83 known COVID-positive samples...

... most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism Engineered...from Staphylococcus aureus ) has a coding sequence ~1 kb shorter than SpCas9 while retaining the same basic...lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...efficiency and fidelity. Nature Communications , 13 (1), 1425. PMID: 35301321 Edraki, A., Mir, A., Ibraheim...interrogation by SpRY-Cas9. Nature Communications , 15 (1), 3663. PMID: 38688943 Hsu, P. D., Scott, D. A., Weinstein...increase its specificity. Nature Communications , 9 (1). PMID: 30082838 Maddalo, D., Manchado, E., Concepcion...CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa...