...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 ...

...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...

...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42....gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...standard curves, and normal controls must be run in triplicate.  The nice thing about qPCR is that since it ... of reactions, it does not need to be run in triplicates. ddPCR is an extremely accurate application, ...

...relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used...in primer Yes Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from...show multiple peaks.    (a) (b)  (c)   Fig. 1: Melt curves showsing (a) a single peak, indicating...changes in ΔΔCT are represented by RQs between 0 and 1. Flipping your experimental and control values during...see this fold change as a positive difference (RQ>1), which may be easier to conceptualize. Just remember... in Molecular Biology, vol. 1160, DOI 10.1007/978-1-4939-0733-5_3, © Springer Science+Business Media New... application, Bioinformatics, Volume 27, Issue 7, 1 April 2011, Pages 1019–1020, https://doi.org/10.1093...

...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ...

...repeats (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional... it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293...-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV ...

...Biotechnology, 10.1038/s41587-023-01756-1. https://doi.org/10.1038/s41587-023-01756-1. PMID: 37095348. Breaking...incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV TAT, Myc and...expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under... membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038/s41592-023-02085-6. PMID...StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587-023-02018-w. PMID...broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023-38582-7...generated a series of StayGold mutants and identified a triple mutant, Y187F, R144I and T155Q, which they named...

...membrane targetin sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. ...Reports. 2020. https://doi.org/10.1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few... fluorescent proteins, they found that adding a triple nuclear localization signal or including fusions...CRISPRi-based synthetic oscillator, bistable network, stripe pattern-forming incoherent feed-forward loop. ...

...incubation of 1° and 2° Must use different species and/or different IgG subclasses of 1° for multi-color...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit and -...dyes are also commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox...NIH’s Developmental Studies Hybridoma Bank (DSHB). 1 ml of antibody-containing hybridoma supernatant is...with the desired fluorophores. In figure 4B below, triple colocalization is possible even when staining cells...

...translation (Figure 1): A tRNA that recognizes a specific ‘open’ codon and utilizes this triplet to incorporate...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...modification allowed the deletion of release factor 1, the protein responsible for terminating translation...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...