... Viral Vector Packaging Service , Addgene is distributing ready-to-use viral preparations in engineered...

... final product should be translated as a single string of amino acids that preserves the sequence...

...Nature . 520(7546):186-91. PMID: 25830891 Savic D, Partridge EC, Newberry KM, Smith SB, Meadows SK, Roberts...

... antibody manufacturer for wash recommendations. Tris-buffered saline (TBS) or phosphate-buffered saline...abundant protein if you’re stripping, since you’ll lose protein with every strip cycle. Troubleshooting the...Increase volume of wash buffer Incomplete stripping (If stripping/reprobing) Check for antibody signaling...signaling after each round of stripping* Use a harsher stripping buffer Too little signal Large proteins...Western blots can be tricky to get right. If you’ve thoughtfully done the technical design of your blot...need such precise measurements, you can use a colorimetric ladder, visible to the naked eye; a fluorescent... choose from.  Transfer Transfers are a little trickier than gels and may require multiple runs to optimize...

...e0124958. LOC8-ing mitochondrial fusion with a new tri-chromatic assay Georg Ramm’s lab at Monash University...cumbersome. To overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or...modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT contains...gene-specific forward primer containing the desired restriction site (the MCS has several options) and a pUCX-specific...resistance cassette, a ribosome binding site, SacI restriction site and a linker region. The PCR product is ...BMC Molecular Biology. 2013 August 20th; 14(1). TRICK: A method for visualizing the first round of translation...the protein product. The system, cleverly termed TRICK, for Translation RNA Imaging by Coat protein Knock-off...

...antibody. TRIM21, an E3 ubiquitin ligase, is the basis of TRIM-away. In normal physiology, TRIM21 recruits...endogenous TRIM21 is sufficient for TRIM-Away, and you only need to deliver the target antibody. TRIM-Away ...Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation Article contributed by Mary Gearing... proteasome function. TRIM-away compared favorably to drug inhibition, with TRIM-away targeting of mTOR.... To assess TRIM21 toxicity, Clift et al. created stable cell lines overexpressing TRIM21, finding that...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly ... methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein and the...

...journey. Figure 2: Annual distribution of items to countries with a distributor. Arrows indicate the year...amazing 111 countries (Figure 1)! But while we were making the materials available to many countries, import...country to country.   Figure 1: Number of countries Addgene distributes to per year, 2005-2023. We identified... we identified countries that would benefit from a third-party “helper”: a distributor who, among other... India, and Mexico. Table 2: Addgene distributors Distributor Partnership Start Date Country Served...in material distribution after we partnered with them (Figure 2). In others, the distributor helped solve...that we could identify the need for distributors in some countries. ‌We’ve developed systems to allow ...

...a sequence recognized by the restriction enzyme MstII. When a restriction digest with MstII and southern...than real-time PCR (4). Restriction mapping and modern sequencing Restriction mapping informs on DNA sequence...different restriction enzyme digests. Incubating a DNA sample of unknown sequence with restriction enzymes...has changed, restriction enzymes are certainly not dead yet! Labs still keep restriction enzymes on hand...This post was contributed by guest blogger, Krissy Lyon, a PhD candidate in Neuroscience at Harvard University...three historical techniques: southern blotting, restriction mapping, and sequencing gels, as well as their...fluorescence for fluorescently labeled probes. Colorimetric and luminescence based detection methods are...

...categories: parametric and non-parametric. In this blog we will be discussing parametric tests. Parametric tests...be met for a parametric test are as follows:   Parametric test assumptions: Distribution – Normal Variables...   Fig. 2: Examples of data distribution profiles.   Parametric tests  Statistical tests fall into...derived from a theoretical distribution. If your data is normally distributed, it will form (more or less... If your data does not fit parametric assumptions, then a non-parametric test is a better option. Stay...numerical risk allowance in a statistical test. Distribution - spread of data; typically, normal (bell curve...data doesn’t fit the requirements above, a non-parametric test may be the way to go, which we will discuss...

...top ten countries for each group (Figure 4).    Figure 4: Addgene deposits and distribution by country...deposited by each country. Countries shown are top 10 depositing countries. (B) Total CRISPR plasmids ...requested by each country. Countries shown are the top 10 requesting countries. FR=France; KR=South Korea...in ~40 different countries, and we’ve shipped CRISPR plasmids to labs in ~100 countries.  The impact Addgene...repository has grown to include many CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative...cumulative numbers of CRISPR plasmid distribution and deposits (Figures 1A and 1B); the cumulative number...cumulative number of CRISPR libraries Addgene has distributed (1C), from 2012 to 2023. Addgene’s collection...

...early-phase clinical trials and have shown promising preliminary results (Dimitri et al). CRISPR engineering...editor-based clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for SCD in ... This ongoing trial is sponsored by Excision Biotherapeutics. Additional ongoing trials The CRISPR therapies...approved or have made significant progress in clinical trials.     Figure 1: Types of delivery and editing...of the cancer-fighting component, CAR. Clinical trials are underway to explore CRISPR’s capacity for both... returning them to the patient. The first human trial involving CRISPR Cas9 for these diseases was sponsored...and CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients ...

...assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site for...fusing the upstream restriction site to the forward primer and the downstream restriction site to the reverse...discussed in our restriction cloning post. However, even more than with standard restriction cloning, it is...of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that...itself are very similar to those outlined in our restriction cloning post with a few quirks specific to the...amplified (usually 18-21bp) When selecting restriction sites, you should use a DNA analysis tool, such...Sequence Analyzer, to allow you to identify which restriction sites are present in a given sequence. You want...

...anticipated restriction analysis results (left) and validation PCR followed by Restriction Fragment Length...This post was contributed by guest blogger, Kristian Laursen from Cornell University. Site directed mutagenesis...conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to...the plasmid template (blue), and introduce a restriction site “A*” (in addition to the “A” site already...in the parent vector). Also shown are the “B” restriction sites, which flank the targeted region. Upon ...amplification, the template is removed by DpnI restriction digestion leaving behind only the mutagenized...Polymorphism (RFLP) analysis (right). Introduction of restriction site “A*” results in an additional band when ...

... tools created by the Trimmer Lab that will be the first antibodies distributed by Addgene.  The plasmids... constructs. The Trimmer lab found that the recognition sequence for the restriction enzyme BciVI was ...Neuroscience AntiBody Open Resource). The antibodies distributed through NABOR are recombinant antibodies expressed...constructing the plasmids for expression of mAbs, the Trimmer lab isolated RNA from cryopreserved hybridomas,...Constructs were then verified by both diagnostic restriction digest and colony PCR.   Fig. 1: (A) ...can be a threat to future reproducibility. The Trimmer Lab found that in addition to recovering RNA from...lab’s entire hybridoma collection. Instead, the Trimmer Lab moved to direct functional validation by immunofluorescence-immunocytochemistry...

..., Vibrio cholerae, Xanthomonas campestris pv. campestris, X. campestris pv. malvacearum, Zymomonas mobilis...plasmid does not have many restriction enzyme sites. Hosts often have a restriction barrier as a defense mechanism...use of their restriction enzymes (Meyer et al. 1977). So if a plasmid has few restriction sites, it may...Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas...Shewanella spp., Thiobacillus spp., Xanthomonas campestris IncQ RSF1010, R300B, R1162 Gram-negative...Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans, Pasteurella multocida, Porphyromonas gingivalis...salmonicida, Yersinia enterocolitica, Xanthomonas campestris, X. maltophilia IncW pSa, pR388 Gram-...

... of an “electricity aggregation program” for all local households and businesses. Electricity aggregation...Addgenies swapping out printer cartridges can place the spent cartridge in the large cardboard box in ...September all will be powered by 100% renewable electricity! A (not so) shocking transition  In early July...program offers options of between 14% - 100% electricity supplied from renewable sources and we have chosen... As a result, we will be drawing 100% of our electricity from renewable energy projects right here in ...- the majority of which are solar, wind, or hydroelectric in nature. The operation of -80C and -20C freezer...save energy. The transition to 100% renewable electricity will allow us to maintain these particularly ...

... mCherry 587nm/ 610 nm human β-Actin Actin 4 Citrine 516nm/ 529nm human α-Tubulin Tubulin Repeatable...principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules...Modules are assembled one at a time by standard restriction enzyme digestion followed by ligation. Upon ligation...the repeatability of the method. Note that the restriction sites of the MCS are only regenerated at the ... 3’ termini of the ligation product, and the restriction site between the modules is eliminated upon ligation... using these techniques are free of specific restriction enzyme recognition sites. Thus, chaining methods...Technology Pros Cons References Golden Gate Type II restriction enzymes Can assemble up to 10 modules in a single...

...unwanted selection markers. Restriction modification systems Because restriction modification systems consist...toxin-antitoxin roles. Restriction modification systems are composed of restriction endonucleases and methyltransferases...preventing the cognate restriction enzyme from cleaving the DNA. A role for restriction endonucleases in plasmid...methyltransferases. Restriction endonucleases cleave DNA at or near specific nucleotide sequences. To ...Escherichia coli in 1995 when introduction of restriction-modification systems onto plasmids allowed cell...these plasmids as compared to those without the restriction modification systems. As with the other toxin-antitoxin...pair is more stable than the other. Here, the restriction endonuclease is more stable than the methyltransferase...

...ApoB-LPs through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman...retroviral mouse CRISPR knockout library Article contributed by Alycia Bittner CRISPR pooled libraries are...for measuring ApoB bound lipoproteins Article contributed by Leah Schwiesow A leading cause of death worldwide...ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents (what is commonly called...called bad cholesterol). However, size distribution and concentration of ApoB-LPs has been shown to have...in vivo. The LipoGlo system represents a new, intriguing approach to expand our understanding of ApoB-...transcription for CRISPR activation in bacteria Article contributed by Will Arnold Baojun Wang’s lab recently developed...

...accelerate your research.   Why distribute virus? Plasmid distribution is booming, but, over the years... we can help them share and distribute other reagents. Virus distribution came up time and time again ...Addgene! As part of our new Viral Service, we’re distributing lentivirus (with many CRISPR tools included ...with light will be available soon. We’re also distributing AAV preps with particular serotypes that can...here. If you have virus expertise you’d like to contribute to the blog, please email us and we’ll get you...quality control data for each of the materials we distribute. Quality control info can be found on each viral... the Viral Service or any of the materials we distribute, please send an email to [email protected]. Your...

...likely to be positively impacted by the trial. During clinical trials, expansion of participants based on ...This post was contributed by guest blogger Subhadra Jayaraman, a doctoral candidate at Binghamton University...surveillance. Dormancy of certain cancers can be attributed to the concept of immunoediting, by way of favorably...TGFβ. T-regs are suppressive immune cells that restrict the production and activity of other effector ...endothelial growth factors (VEGF) in tumors is attributed to a signaling cascade, where lack of oxygen ...therapies include radiation therapy (owing to contributions from Madame Curie), chemotherapy, and surgical...leukemia and lymphoma and are in their clinical trial phase. In all of the above therapeutics, techniques...

...encouraging user contributions (using simple web forms) while also maintaining a relatively strict database design...This post was contributed by guest blogger Talley Lambert, a Research Associate at Harvard Medical School...design of an imaging experiment.  The choice is non-trivial, however, as FPs are tremendously complicated entities...on a web-based interactive visualization of FP attributes (which turned into www.fpvis.org).  That page...and we very much appreciate even those small contributions). Of course, no community-editable database ...simplifies the process of building advanced multi-parametric search queries.  Looking for a bright, monomeric...frequently) for the sake of comparison or ease of retrieval.  These collection pages can be kept private, ...

...their destination co- or post-translationally. TRICK: Visualizing the first round of translation While... co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging by Coat protein Knock-off...RNA-binding proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change...RNAs appear red. To enable this color switching, TRICK requires the following components: a GFP protein...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important...important in development, and Halstead et al. used TRICK to examine translation of Drosophila patterning ...patterning gene oskar. Early in development, the osk-TRICK reporter was double labeled, indicating translational...

...fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside...which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have...They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on....that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment.... If you inserted these two pieces of DNA by restriction enzyme cloning, you would have to do it in two...the smaller overlapping sequences left with a restriction digest. A study in 2013 found that Gibson assembly...PCR step difficult--OR there is no convenient restriction site for linearization. This is a perfect case...

...permission to distribute. Distribute pending QC: your plasmids become available for distribution as soon as...archive any plasmids you've deposited with us and distribute them to scientists worldwide. What's more, depositing... pre-publication plasmids" and set a desired distribution status. You can submit "Published plasmids":...description of the deposit. Select your desired distribution status: Hold for publication: we will initiate...additional questions. Bacterial Strain Addgene can distribute your bacterial strains if they: are E. coli ... +1 (617)-ADDGENE (233-4363) if you’d like to distribute your bacterial strain(s) through Addgene. Description...information for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should ...

...that plasmid deposits come from 49 countries* and reached 102 countries. We started from 474 plasmid deposits...deposits coming from two new countries bringing the total to 51 countries with plasmid deposits!  ...Twitter!  That’s today through Friday. There will be trivia, prizes, and more! We wanted to take a week to ...impact in open science. Depositors consistently contribute to our collection so that we can offer the scientific...been requested about 1,500 times from labs in 45 countries*. While the bulk of these plasmid deposits came...you are a depositor or not. We will have Addgene trivia (with prizes!), polls, and shout outs, and we’ll...the schedule for the week: Monday: Fun stats and trivia begins! Tuesday: Depositor features (plasmid deposits...

...Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  Listen to the anti-CRISPR podcast...fluorescent voltage indicator Archon Article contributed by Shreya Vedantam  Listen to the voltage indicator...segment The neurons in the brain compute using electrical impulses to coordinate emotions, thoughts, and... behavior. Historically, scientists study the electrical activity in the brain by inserting electrodes...thousands of proteins for one that can report electrical activity via imaging. They took the light-sensitive...easier with a 2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning...pUC57-L3L2) that offer the advantage of using restriction digest cloning to introduce the GOIs into the...

...anode/iblot gel matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper...remain intact and the protein is separated through a matrix by charge. In a denaturing western, the protein...smaller molecules moving more quickly through the matrix. For the purpose of this blog, we will be focusing...confirm the size of the protein of interest. An electric current applied to the gel causes the negatively...proteins encounter less resistance in the acrylamide matrix and migrate faster through the gel. For better ... In addition, PVDF membranes can be repeatedly stripped of an antibody and reprobed with different antibodies...sandwiched” together with blotting paper and an electric current is applied causing proteins to migrate...

...fundamental steps: creation of the RNAi trigger delivering the RNAi trigger characterizing the RNAi phenotype...This post was contributed by guest blogger, Robert Orr, who recently received a Ph.D. in Biology and ...complementary to your gene or genes of interest to trigger RNAi activation. A typical RNAi experiment can ...RNAi-based investigation. Creation of the RNAi trigger requires a few initial considerations, such as ...artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent method due to ease of use...assortment of siRNAs generated from a long hpRNA trigger, only a single ~21-nt interfering RNA is produced...precursor mentioned above and is a popular RNAi trigger in the mammalian research community. Fortunately...

...Comparison Between Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning...This post was contributed by guest blogger Meghan Monroy, a graduate student in Protein Science at the...elements of biomedical research. Traditional restriction cloning is one of the oldest of these techniques...purification, ligation, and transformation. While restriction cloning is still routinely performed by many ...Cloning Choose Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - ...products and digest with methylation sensitive restriction enzyme DpnI to remove the templates. Digestion... in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis....

...three tabs.  Each of the three tabs - Contributions, Indel Distribution and Traces - provides particular details...analysis screen. ICE analysis details Contributions   The Contributions tab shows the inferred sequences present...the most 5’ cut site.     Indel Distribution In the Indel Distribution tab, you’ll find an Indel plot ...This article was contributed by Jessica Roginsky, Scientific Support Lead at Synthego. Article source... useful measure to determine how many of the contributing indels are likely to result in a functional ... interested in understanding how many of the contributing indels are likely to result in a functional ...plot (left panel) which displays the inferred distribution of indel sizes in the entire edited population...

...researchers in 45 different countries — and as of 2024, we’ve distributed over 100,000 viral vector preps...adeno-associated viruses (AAVs), will know just how tricky these tools can be to use. AAVs, which can target...repository each year from 2016–2023. B) AAV preps distributed per year by Addgene from 2016–2023. Tools...requested (Figure 3). Over 400 biosensors were distributed in 2023 alone. You’ll also find a large number...from 2016–2023 by type of tool. B) AAV preps distributed by Addgene per year from 2016–2023 by type of...AAV preps for the seven most popular serotypes distributed per year by Addgene from 2016–2023. How to... A) GCaMP6, jGCaMP7, and jGCaMP8 viral preps distributed per year compared to other AAVs preps from 2016...

...attL-containing entry vector.   Method C: Restriction cloning of a restriction enzyme fragment containing the DNA...scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can choose a molecular...Figure 5: Method C to create an entry clone: Restriction cloning the inset into an attL-containing entry...integrity of your expression clone via sequencing or restriction digest! Then, you can transform or transfect ... day, as opposed to 2+ days with traditional restriction and ligation cloning. It is also possible to ...vectors. The cloning process is simple -  no restriction, ligation or gel purification steps are required... generate entry clones by TOPO cloning or by Restriction Cloning Destination vector attR sites for recombination...

...James Trimmer of the University of California Davis School of Medicine on this grant. Dr. Trimmer has created... ready-to-use-proteins to facilitate use. Dr. Trimmer’s lab will ensure the quality of the antibodies ...Addgene to bring this resource to life,” says Dr. Trimmer. But in order to create such a resource, we need...on being financially self-sustaining through distributions of research materials, such as plasmids and ...new project to reach a sufficient number of distributions to cover costs, which can limit expansion into...Initiative has been funding laboratories, such as Dr. Trimmer’s lab, to create recombinant antibodies for use ...https://doi.org/10.1038/520295b Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson ...

...Deposit and Distribution Agreement establishes the terms under which your material will be distributed. In signing... of distributing under Addgene’s Industry MTA (IMTA), which allows the plasmids to be distributed for ... 784,795 MTAs with 8,798 organizations in 111 countries. From 2019 to 2023, we averaged facilitating 89,477...actually own the majority of the materials we distribute; therefore we are not a party (signatory) to ...re not! The vast majority of our plasmids are distributed under the UBMTA, which is widely used across ...organization agrees to let Addgene receive, store, and distribute your material under the specified terms. This...depositing institute that decides the terms of distribution for their plasmids. MTAs at Addgene Since Addgene...

...This post was contributed by guest blogger Nathaniel Roquet, a PhD student in the Harvard Biophysics ...Once expression of a particular recombinase is triggered, its effects on the state cannot be undone and...predominant state of registers within a sample using a metric that we describe in the Materials and Methods section...only semi-quantitative. It won’t tell you the distribution of states in a sample; instead, it will suggest... of registers in a sample based on a similarity metric that we defined in our paper. In our opinion, the...attB-attP pairs on a register to record all permuted substrings of a set of chemicals. Specific examples of arrangements...arrangements that should be able to record all permuted substrings of up to seven chemical inputs are given in Table...

...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, ...to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble... Gate protocols, complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet...destination vector contain multiple internal restriction sites that may not be amenable to "domestication...cloned DNA as tandem multimers using class-IIS restriction enzymes. Lee JH, Skowron PM, Rutkowska SM, Hong...

...unique restriction enzyme site and unique primer sites for more rapid screening by restriction digest ...for genetic optimization of industrial yeast strains. However, industrial yeast strains are more difficult...CRISPR library Addgene has had the privilege of distributing several human and murine CRISPR pooled libraries...single-tube, 3 day process by utilizing pre-assembled trimers to reduce the number of cloning steps. The FusX...PMID: 26787909 EasyClone 2.0 Yeast Toolkit Many industries, such as the food and beverage industry, the ...transform. To increase the genetic toolbox for industrial yeast strains, the Borodina Lab has recently ...et al. Nat. Methods 2009. PubMed PMID: 19915560 TRIBE: Targets of RNA-binding proteins identified by editing...

... on versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to ... But...how? Cloning is no longer limited to restriction enzymes - although many labs still love using...your E. coli do the work for you. Traditional restriction enzyme and ligase based cloning is a relatively...The ligase cost is comparable to that of the restriction enzymes and overall you’re looking at a price...of a few dollars per reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning...appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations. The...efficient due to the use of the versatile type II restriction enzymes. In Golden Gate cloning, the desired ...

...Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole field ...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...would take another 6 years before the 1st clinical trial started, and 10 more years to obtain FDA approval...natural serotypes are currently used in clinical trials, a variety of engineered ones have shown great ...in preclinical studies and could reach clinical trials soon. The AAV safety profile is excellent and ... cells using a lentiviral vector and a clinical trial is ongoing. Moving forward with ocular gene therapy...AAV-based ocular gene therapies is impressive: clinical trials for choroideremia, X-linked retinitis pigmentosa...

...This post was contributed by guest blogger Katrin Michel. Cre-lox is an incredibly popular and powerful...times. Many thanks to our guest blogger, Katrin Michel! Katrin Michel is a Postdoctoral Researcher at MIT...pair palindromic repeats separated by an 8 bp asymmetric core. Recombination between these sites can lead...discovered that it is not the exact sequence of the asymmetric target site core but its 8 bp length that is ...plasmids and the low concentration of recombinase restricts their expression to individual cells. With this...knockout in the same mouse.   Scientists always strive for more. In order to analyze the function of individual...Through a partnership with genOway, we are able to distribute materials containing FLEx technology. Read the...

...level, read on for more in-depth tips on probe chemistries, primer optimization, controls, and analysis....Picking your chemistry  The two most common qPCR chemistries are hydrolysis probes (which you probably know...commercial name: Sybr). However, there are other chemistries available and they can be the more optimal choices...choices at times. For example, chemistries that rely on hairpin design of probes/primers often offer higher...stable than the hairpin bond.  Alternatively, chemistries that combine the primer and probe into a single...comparison of features of several commonly used chemistries)   Chemistry Background noise Probe ...gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design...

...studying the brain in a way that goes beyond such restrictions, you may want to consider a systemic capsid ...enhanced function after intravenous administration, distributing widely throughout the organism to target large...and recombination target sequences, denoted by triangles] facilitate tissue or cell type specificity. An...ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal ...your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb) for your...promoters and enhancers (Mich et al., 2021) can help restrict expression to your cell type of interest, while... with Cre-expressing transgenic mouse lines to restrict transgene expression to specific tissues and cell...

... a SmaI or XmaI restriction enzyme digest (the ITRs contain SmaI and XmaI restriction sites). Another ...these tools, I found myself writing AAV production tricks into the margins of my protocol. While these tips...recombination of duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary...ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of different concentrations...during AAV production is overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected...day. Refer to the AAV Iodixanol Gradient Ultracentrifugation Protocol for more details. Pro tips Watch...titer AAV. Do you have any AAV production tips or tricks? Please share them in the comments below! Special...

...This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such...limitations through the use of engineered Type IIS restriction enzymes and careful choice of junction sequences...involves the simultaneous activities of a Type IIS restriction enzyme such as BsaI, and a DNA ligase such as...receives the assembly after similar Type IIS restriction enzyme cutting results in 5’ four base overhangs... provide optimal reaction temps for both the restriction enzyme and ligase, thereby enhancing the ordered...destination construct and functioning Type IIS restriction enzyme and DNA ligase, any single omission should...

... This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means... assumed that the molecules are homogenously distributed throughout the volume and can freely tumble in...be much higher than if they are homogenously distributed and mobile throughout the cell. If two copies...CFP, YFP, BFP, Cerulean, Turquoise, Venus, and Citrine) can be monomerized with an A206K mutation [2].... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing the lowest...

...research by banking and distributing monoclonal antibodies at cost. We distribute over 65,000 antibody units...The following post was contributed by the Developmental Studies Hybridoma Bank. Antibodies are the most..., is a repository for monoclonal antibodies, distributing over 3,000 antibodies and associated hybridoma...the University of Iowa. The minimal fees from distribution funds our operation. Antibodies are glycoproteins...PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies against popular fluorescent...Posts about Scientific Sharing: The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years...

... media platforms are available or used in all countries; not all areas of any given country have equal...can guarantee you that either resource or time restrictions (or both!) will be a major limiting factor at...flexibility to visualize and contextualize. Can be distributed in digital or analog media formats. Cognitively...platforms can splinter audience. Primarily digital distribution (though radio is possible.) Written works...works Low Flexible. Low barrier to entry. Ample distribution channels both digitally and physically. Easy...overwhelming your audience. Digital or print distribution.  Static. Can be difficult to convey an entire...highlighting, or omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific...

...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes...system (CNS). To address this technological gap, a trio of recent papers describe the development of CRISPR...method to dissect the individual and shared contributions of transcript isoforms to neuronal physiology...surface protein for which there are good flow cytometric antibodies, downstream of the TUJ1 coding sequence...these cell types are used. The lack of good flow cytometric antibodies for many CNS-resident cell types (...

...This post was contributed by Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...no one is suggesting all data be stored in a retrievable format, we too rarely preserve “raw” data of ... methodological detail. Publishers have also contributed to this issue by requiring ever more condensed...resources can offload the burden of data storage and distribution. I would argue that the journals themselves ...journal repository, they are likely too large to be retrieved by others. Other new data initiatives are also...and understand their data. Likewise, we want to contribute to helping others understand ours. Improving ...