...filled depends on cellular factors. Multiple labs tried to identify beneficial factors to include in even...bases. Thus, base editors have an activity window restricted to the stretch of ssDNA created by Cas9 binding...

...There is something akin to a frenzy as the crowd tries to weigh and determine the value of every sweet ...This post was contributed by guest blogger Cameron MacPherson at the Institut Pasteur CRISPR software...falciparum (the parasite that causes malaria). I attribute their success to technical expertise, thoughtful...Cas-OFFinder provides a bare-bones data dump of ALL strings in a genome using any IUPAC encoded pattern at ...biotechnology(2014). PubMed PMID: 24880488. 2. Hsu, Patrick D., et al. "DNA targeting specificity of RNA-guided...

... 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...