...identical molecules of DNA. Gene targeting takes advantage of this natural process to replace a targeted ...marker HSV-tk is used to select against random recombinants.   Design your targeting construct. For...been cloned into the vector on either side of an antibiotic resistance gene. To avoid selecting for cells...will first perform a positive selection for the antibiotic resistance gene and then a counter selection ...Remove the positive selection marker. Since an antibiotic resistance gene can affect a cell's phenotype...a true knockout can be lethal, and you'd instead want to create a conditional knockout. To make a conditional...

... between the microhomologies.MMEJ has certain advantages over HDR. Some species don’t have good HDR systems...locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus...into the FBL locus. Upon sequencing of puromycin resistant clones, they found that 80% and 50% of clones ...technique can be adapted to other transgenes. It’s important to note that the fluorescence level observed will...follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes into AAVS1, the...to insert cDNA constructs into this locus. One important question to ask is how the efficiency of PITCh-based...

... expressed proteins. Here we will review what advantages degron tags can offer over other protein control... protein expression. These tags offer several advantages over the alternative systems – CRISPR, siRNA,...function, the SMASh tag self-cleaves itself when you want the protein around, leaving a minimal additional...with the most alternative functions – there are antibodies to it and ways to achieve fluorescence with it...might be the way to go (dTAG/SMASh). If you don’t want to deal with additional components beyond the minimum...Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...

... cells. To study this phenomenon you will likely want to monitor the voltage change across the membrane...comparable to its one component counterpart and is significantly brighter and available with red-shifted fluorophores...for penetrating deeper tissues depths (1 mm), important for certain applications such as in vivo tissue...encoded component of the system to offer unique advantages depending on your experimental system.   ...and this voltage is particularly physiologically relevant in neurons, myocytes, and endocrine cells. It ...sensor selectively is especially essential. If you want to learn more about how viruses can be used for ...

... to achieve: Do you want to permanently knock out or knock-in a gene? Do you want to activate or repress...trying to create a single point mutation? Do you want to create a fusion with a reporter protein such ...sufficient? Which cell types will you be editing? Do you want to deliver the components as DNA, or would mRNA ...and Cas9 are required for any application. If you want to include a homology directed repair (HDR) template...well characterized in terms of CRISPR use, you will want to invest some time in optimizing and testing the...

...vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives? Then do so! It's a tremendously...that anyone else who came up with the same idea or wanted to build a gene drive in a model organism would...experiments2,3. While that didn't work quite as well as anticipated4, greater awareness will certainly improve safety...regions that don't have resident populations of the relevant mosquito species. Reproductive confinement involves... cas9 gene). An immunizing reversal drive is a variant that also spreads through the wild population and...gene drives. Science 345, 626–628 (2014). PubMed. Gantz, V. M. & Bier, E. The mutagenic chain reaction: ...

...think of themselves as “scientists.” We had some accountants running our math tables!  Be realistic about...exposure. If you expect a lot of kids, you’re going to want a lot of activities (try for a dozen!). The best...presentation because, in my opinion, it was more important to communicate the applications of CRISPR over...use it? This last section was arguably the most important, and it was a little worrisome for me since bioethics... next year. There are many reasons why you might want to reach out to your school community as a scientist...scientist. Perhaps you have young children and you want to make sure their STEM education is supplemented...

...choose? Which way is the right way?  First of all, I want to put to rest a common misconception. I find personal...natural - if it works so well for you, of course you want to share it with others! After all, won't it work... to where your audience already is.  You’ll also want to keep in mind that access and availability to ...resources do you have?  No matter the project, I can guarantee you that either resource or time restrictions ... design a hands-on interactive activity using elephants than it is to design one demonstrating mitosis...Consider also the amount and type of information you want to convey. Small tidbits and fast/fun facts are ...

... likely seem important for any virus, we will see that they are very central determinants of how AdVs ...toward saying that host-virus interactions are an important part of the biology of adenoviruses (AdVs). AdVs...from the genomic plasmid can be used to house a recombinant sequence (transgene) instead.    Although the...natural AdVs, replication-competent viruses with a recombinant genome, and adenoviral vectors all have a genomic...basic research, like other viral vectors, though significant challenges remain (Wold & Toth, 2013).  While...

...Addgene's Antibody Guide Addgene's Ready-to-Use Antibody collection Addgene's Recombinant Antibody Plasmids...sure that the sample is pure and free of any contaminants that could affect the measurements. You'll need...below 0.95! More resources on the Addgene blog Antibodies 101: ELISAs The Four ELISAs and When to Use Them...

...the cells’ germline genetic variations. They then wanted to identify cancer-associated genetic dependencies...ancestry, Sean and Aaron knew that their study had significant statistical limitations, and expected to find...Cas9 from cutting, both of which can introduce significant bias into the results. But the reference genome...design of the experiment or dataset itself. It is important for scientists to not only be aware of the potential...ancestral bias and the human genome Wang, T., Antonacci-Fulton, L., Howe, K. et al. The Human Pangenome...doi.org/10.1038/s41586-022-04601-8 Lewis, Anna CF, Santiago J. Molina, Paul S. Appelbaum, Bege Dauda, Anna...

...). Transformed bacteria are then grown up on antibiotics such that only bacteria with the plasmid can ...filtered pipet tips and prepare fresh media with antibiotics at each stage. The barcodes and LIMS help us ...enter the cells. Some samples require a reduced antibiotic concentration or need to be transformed into ...tubes. 1D barcodes are better for tubes that are meant to be read one at a time. 1D barcodes can be purchased...misread.  2. Track the container - It is just as important to track the location of the container housing...to fulfill orders.  The barcoded tubes help to guarantee that no samples get lost during their journey....

...systems. Can you describe this technology and its advantages? Fujii: In the last several years, my lab has...is being involved in the scientific community important to you and your lab? Fujii: I first knew Addgene...considering the peer-review system of journals and grant applications, for example. However, at the same ...interact with each other, which is critically important, especially for young researchers. It was a really...living for Japanese, which is not trivial, and the grant situation is currently much better than that in ...tough competition for academic positions and poor grant situation. So, young researchers need to publish...

...recipe. It’s unrealistic to plant, grow, and harvest grains every time you want to bake a cake. Instead, ...development happening at Addgene under the hood. A significant portion of our development may not be visible...changing needs of the teams at Addgene. Another advantage of Scrum is that it provides transparency to everyone... been updated in several years) or they make significant changes that make it incompatible with our software...software from Python 2.7 to Python 3.5. There are significant changes to the language and it took us a total...

... maybe you’re researching a topic for which no relevant or recent review exists. Once you pick a topic...format also has the benefit of exposing you to relevant researchers in the field. Finally, you can sound...from start to finish. Find the information that’s relevant to you, note it, and move on. At the beginning...retreat to the woods for months at a time, so take advantage of whatever time you have. If you’re like me, ...were, and you could find the original paper if you wanted to learn more. Another example: we made a schematic...Tips for Improving Your Next Manuscript 6 Tips for Grant Writing Resources on Addgene.org Check out Our...

...targets for complement-based killing by anti-alphaGal antibodies. The VSV-G envelope protein is inactivated...different viral membranes, tropism, and advantages and disadvantages: Table 1 (Joglekar et al., 2017)  Table...its own, it needs to be pseudotyped. This takes advantage of the EnvA-TVA receptor-envelope system (Wickersham...2006) Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat Methods 4:47–49 . https://doi.org...

...speed easier and faster when you’re an active participant in the training. In this blog we will review ...valuable lessons along the way. We think it’s so important that we have written plenty of articles on building...bench mentor-mentee relationship is extremely important – without a good set up it could be very difficult.... Preparation before bench work Are there any relevant papers you can read prior to the start of your...through the protocol mentally (or physically where relevant) and identify any gaps or questions you have, ...everything is magically made up by helpful TAs or assistants, and sitting out on the bench waiting for you...

...between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine bases (cytosine, ... purine to a pyrimidine or vice-versa. Note: hypoxanthine (shown) is the nucleobase component of the nucleoside...nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar. Their fusion of wild-type ...glycosylase / alkyladenine DNA glycosylase  Removes hypoxanthine from DNA, creating an abasic site. The same ...one name. Indel Insertion/deletion A type of unwanted DNA edit. AYBE, AXBE, ACBE Adenine transversion...models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing...

... L., Martínez-Antonio, A., Quiñones-Valles, C., Espíndola-Serna, L., & Martínez-Antonio, A. (2011). Mechanisms...base pairs for the protein to be translated. An antisense RNA called Inc, also encoded by the ColIb-P9 plasmid... the host with a beneficial function (usually antibiotic resistance).  In the lab, low copy plasmids may... competition between pseudoknot formation and antisense RNA binding at a specific RNA site. The EMBO Journal...Leonard, A. C. (2018). Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome...

...Biddle, of Only Good Antibodies, who shared their antibody validation expertise. Antibody Validation for Flow... Caitlin Faulds, who introduced the very first antibody deposit (the Integrin collection) from the Institute...Institute for Protein Innovation. An Integrin Antibody Toolkit from IPI Simon Moe, from Promega, who ...