...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...

...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...

...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....