...An antibody that has been cited thousands of times may be a safer bet than one that has never been used...work. Whenever possible, choose an antibody that has been validated in your specific application. Consider...cross-reacts with. Ideally you will find an antibody that has been validated in the species your protein of interest.... Talk with your peers. If someone in your field has used the antibody before you will not only have peace...Uhlen, 2016). Be sure that the antibody you choose has been validated using one or more of the proposed ...

...targets by: Mike Lacy Adding to their set of anti-HA frankenbodies (Zhao et al. 2019), Tim Stasevich and...iRFP713, SNAP-tag, and HaloTag fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag...Adapted from Liu et al. 2021. Anti-FLAG and anti-HA frankenbodies have a variety of applications, including...blotting, and more. Given the popularity of FLAG and HA tags, you may not even need to clone new constructs...requires a small epitope tag (FLAG is eight amino acids, HA is nine), multiple repeats of the tag can recruit...larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic screens because...

... expressing IgM. IgM has a pentamer structure, which means each IgM molecule has a binding capacity or...function is unknown. It has a monomer structure with a valency of two and has no subclasses. IgG IgG is... appropriate isotype control. An isotype control has the same constant heavy chain domain as your primary...replaces the hinge region in IgM antibodies. IgM has no subclasses and is found mostly in blood. IgD While...

...Service since 2009. He has  developed a proprietary, low cost cloning protocol that he has used for cloning ...least 24 bp with the template and if the sequence has high GC or AT content, increase the overlapping region... most of the troubleshooting regarding this step has to be strategy specific, there are few general parameters...ratio of 1:2 (plasmid:insert), but this parameter has to be optimized according to the strategy adopted...satellite colonies and they form when all the antibiotic has been used. Don’t worry too much, just pick the big...

...communities and welcome suggestions in the comments.  X has moved significantly away from the functionality and...conversation in the scientific community. Addgene has been considering a gradual move off X as a response...conflict with our values. For this reason, our timeline has accelerated. We significantly reduced posting on ...

...Kcleave (min-1). SpCas9 has maximum activity from 35-45 °C, while GeoCas9 has maximum activity from 50...the recent discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR...stability in human plasma The last trick GeoCas9 has up its sleeve is its increased longevity in human...stable when incubated in reaction buffer alone, but has limited activity when incubated in diluted human ...

...You will also have to make sure that your plasmid has been incorporated into the host organism, usually..., c-Flag pcDNA3, comes from the pcDNA series. It has the CMV promoter for mammalian expression, a multiple...mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also upgrade your basic mammalian expression...considered to be a more robust expression system and has its advantages; however, remember that you will need...the specific mutations. For these purposes Addgene has a large collection of yeast expression vectors with...

...sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting ...genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose. Epigenetics began...many TALEN-based technologies, the advent of CRISPR has made targeting much easier! With the help of guide...modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including...BioRender.com. Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation...Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš’ lab has deposited pdCas9-DNMT3A-EGFP and pdCas9-DNMT3A-PuroR...DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion...

...until 2005 [1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The...offers a large cargo-carrying capacity (over 200 kb has been demonstrated) with no known upper limit [1]....particularly useful when the gene being expressed has threshold effects, such as with signaling molecules...transgenic cell line with the piggyBac™ system is that it has a preference for integration at regions of open chromatin...removal, which alters the endogenous DNA sequence and has been suggested to cause cytotoxicity [5]. Second,...efficiency. While the efficiency of excision with PBx has been shown to be higher than that of the wildtype...Cre recombinase, the PBx is highly specific, and has not been shown to cause genomic abnormalities when...

...mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector that can be ...and the subsequent biochemical characterization, has enabled scientists to engineer a wide variety of ...green to red when it is oxidized. The Timer tool has already been used by several groups to monitor protein...imported to the mitochondrial matrix. Gottlieb’s lab has used an inducible MitoTimer reporter using a Tet-... for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking....optimization through engineering, Ed Boyden’s lab has identified a variety of new channelrhodopsins for...nicknamed Chronos, is blue and green light-excitable and has faster kinetics than those of other channelrhodopsins...

...between a cell that has had one viral particle integrated into its genome and a cell that has had multiple integration...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected... be significantly higher than the same prep that has been stored in the freezer. If you plan to freeze..., you may want to consider using an aliquot that has been subjected to a freeze/thaw cycle for titering...CRISPR Genome-Wide Screening Learn How Lentivirus Has Been Used for Lineage Tracing Additional Resources...

...science has become increasingly restricted to academic and industrial labs, but recently there has been ...KittyBiome has been incorporated as a non-profit entity and so far most of their funding has come from ... the predictions about the effects this mutation has on locomotion in our computer model to the effects...Genetics Center (CGC) at the University of Minnesota has declared our makerspace an educational institution...

...to define how you will know that the relationship has been successful – what are the desired outcomes? ... Dora Farkas, founder of Finish Your Thesis (who has also blogged for us) offers a free report on her ...called “Secrets to Success in Graduate School”. She has also launched an online career coaching program that...in Madison, Wisconsin she also works remotely and has experience as a scientific recruiter. Chris Humphrey...of From PhD to Life. She runs great tweet chats, has an outstanding blog and, while her PhD is in History...remotely, will travel for speaking engagements and has many areas of specialty from leadership development...

...also the fastest test that has been developed so far.  A similar method has been optimized to detect the...Two-step-SHERLOCK has a sensitivity in the zeptomolar range while One-step-SHERLOCK has a sensitivity in...State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...full-fledged pandemic. Containing the spread of this virus has become an utmost priority for countries around the...the outbreak ultimately flattening the curve, it has become evident that widespread testing is crucial... 83 known COVID-positive samples tested, DETECTR has a positive prediction rate of 95% and a 100% negative...

...in living cells. The HA frankenbody is an HA tag detection probe. It contains an HA-binding scFv that’s...often genetically fused to other proteins, which has led to their application for basic and translational...fluorescence intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged...s fused to a fluorescent protein such as GFP. The HA frankenbody works just like an antibody-based probe...of cytotoxic granules from the T cell. When a CAR has an scFv that binds cancer-related antigens, binding... Addgene blog: Read about frankenbodies, an anti-HA scFv-based probe for fluorescent imaging. Learn more...

... dark but dissociate under blue light. This tool has now been modified by Torsten Wittmann’s lab at UCSF...genetic or pharmacological manipulations on cells, and has potential use for a wide range of applications in...monomeric, 4-5 fold brighter than mNeptune684, and has a favorable pKa of 5.6. To test out this new FP in...The increasing popularity of single base-editors has resulted in the rapid expansion of the genome editing...expansion of genome editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid...

...There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field ...CRISPR Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment...CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients experiencing...desired edit, but this particular class of treatments has quite the opposite aim: to destroy. One treatment...way. Delivery, especially for systemic approaches, has its own unique set of obstacles. Thus far, several...delivery tends to be trafficked to the liver. Thus, it has been used for hATTR therapy, since TTR is primarily...

... It was inspiring to hear how the genome project has already had a major impact on undiagnosed and rare...its work on the origin of cellular life. His lab has long hypothesized that life started with RNA and ...prokaryotic genome. Then he desribed how P. furiosus has systems to defend against both DNA and RNA viruses...technical developments. Using the Tet-on system, his lab has created a tetracycline cas9 inducible knock-out system...in a complicated conformation “not like DNA which has a boring structure.” That certainly summed up this...this conference—nucleic acid science still has many surprises for the researchers in this field. Resources...

...enormously plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to... to create specific edits in DNA, there has also been significant work on constructing Cas9 transcriptional...CRISPR activation for genes with known functions has the potential to be used in therapeutics, as it can...changes in phenotypes. For example, CRISPR activation has been used to differentiate induced pluripotent stem...creation of innovative technologies. To date his group has develop several popular CRISPR-based tools for altering...Genome-wide Screens with CRISPR/Cas9 Learn How CRISPR Has Been Used with AAV Resources on Addgene.org Find...

...Recently, Xue Sherry Gao’s lab at Rice University has developed DAP arrays for efficient multiplex base...process and release its own gRNAs from a single array, has been a top choice for multiplex gene knockout or ...proper functions to enable efficient base editing and has yet to be developed for prime editing, both and their..., which is conserved in all living organisms and has previously been used for multiplex Cas9-nuclease ...lengthy promoters, such as U6 or EF1α promoters, has limited the number of gRNAs that can be packaged ...