...biology, technology and application. Viruses. 2011 Jun;3(6):677-713. DOI: 10.3390/v3060677 Varanda C, Félix...clinical settings. AAV is also a cool tool in the laboratory! It is ideal for whole animal administration,... be helpful to you. Interested in learning more about a specific virus? We have you covered with guides..., adenovirus, and AAV. If you’re wondering more about what applications these viruses are good for, we...

... PMID: 14166766. PubMed Central PMCID: PMC300121. 3. Söderberg, Ola, et al. "Direct observation of individual... FUNCAT Professor Schuman’s technique applies metabolic labeling using a noncanonical amino acid, azidohomoalanine...both quantitative and spatio-temporal information about the turnover of specific endogenous proteins in ... interested in neurobiology.  She is passionate about communicating science to all audiences. Right now...

... to wane about a month later, the animal is given additional booster immunizations every 2-3 weeks to ...  Read about another common type of antibody for research, monoclonal antibodies Learn about the advantages...

...PMID: 21364278. PubMed Central PMCID: PMC3069789. 3. Kügler S, Kilic E, Bähr M. Human synapsin 1 gene ...Resources on the Addgene Blog Learn More About Lentivirus Learn more About AAV Get the Scoop on Our Viral Service...

...and best characterized of these is the Src homology 3 (SH3) domain family, known to be involved in cytoskeletal... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.  ...can take as long as several days. In 2015, the laboratory of long-time Addgene depositor Alice Ting used...

... to antibody response 2) very high immunogenicity 3) and long-lasting immune response even after a single...Viruses as Biological Tools blog can help you learn about the different types of viruses and pick one for ...Addgene.org Browse Our Viral Vector Pages Learn About Viral Vector Biosafety Deposit Your Viral Vectors...

...synthetic biology tools for cyanobacteria. Figure 3: Assembly of plasmids using the Cyanogate Kit and ... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.    ...tracing using the transgenic zebrafish model, ubi:zebrabow. In this system, cells express various fluorescent...

...ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as an R-loop....power of dsRNA for a long time, even before we knew about all the diverse in vitro production mechanisms. ...repetitive sequence. All of the R-loops described above play distinct roles in promoting cellular processes...

...10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, extract...they get old, they lose competency). If all the above is correct, you should probably have a second and...blog posts Read blog posts on plasmid cloning Read about different plasmid elements Resources on Addgene.org...

...PubMed PMID: 2839833. PubMed Central PMCID: PMC281709. 3. Tissue– and site–specific recombination in transgenic...discussed below. How does it work? As alluded to above, the Cre recombinase catalyzes the site specific...Combining this with some of the loxP methods described above, a genetic modification can be restricted based ...

...spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other species can recognize...our CRISPR 101 blog posts Find more information about Cas proteins Download the CRISPR 101 eBook Resources...

...transfection. The other (mKate2) is regulated in the 3’ UTR by four miRNA target sites and thereby serves... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.    ...

...sequences more efficient and less risky.   Fig. 3: Development of a cleavage-free dCas9-based gene editor... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here. ...

...protein expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. A: individually... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.  ...

...and complete screening in just 3 days? Thanks to Tyler Jacks’ lab, this 3-day goal is is now attainable...construction of a so-called “genomically recoded organism” (3) but its portability to other bacterial strains remains... the assembly of TALE repeats into a single-tube, 3 day process by utilizing pre-assembled trimers to ...but be sure to let us know if you'd like to write about your plasmids in a future blog post. No time to ...pooled libraries, but thanks to a deposit from the laboratory of Dr. Ji-Long Liu at Oxford University, we are...you plan to use GMAP for your experiments, think about depositing your new plasmids with Addgene to help...and context-dependent human fitness genes, the laboratory of Jason Moffat at the University of Toronto ...

...enter the cells so they can go to work. Figure 3: Inducing the double strand break and introducing ...the Addgene Blog Learn More about Homology Directed Repair Learn More about Non-Homologous End Joining .... For a C-terminal tag, as shown in the example above, we inserted a linker and GFP tag at the end of ... and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone... Plasmids, plasmids everywhere To find out more about gene edited hiPS cell lines generated using the ...line. The editing strategy and design described above can be used to create similar donor plasmids for...microhomology-mediated end-joining (MMEJ). To find out more about gene edited hiPS cell lines generated using these...

...ankyrin repeat proteins. DARPins typically consist of 3-4 repeats, have high specificity and binding affinity... time, when someone speaks about an affinity reagent, they’re talking about a standard antibody: An ~150kD...mutation, allowing diabodies to be used for assembling protein nanostructures. Diabodies readily co-crystalize...for clinical applications, including imaging.  Diabodies are ~60 kDa proteins formed by two Fab fragments...2019). Structural diversity and flexibility of diabodies. Methods. 10.1016/j.ymeth.2018.09.005 Muyldermans...

...Always ask for references and follow up with 3-4 to hear about those clients’ experiences. Good coaches will...Individual Development Plan Learn a Little about The Academic Labor Environment from the Future of Research...research.  It helps to spend some time thinking about what your goals are for the coaching relationship...with someone you connect with. Talk to the coach about their “style” and make sure this is a style that...coaching group is Still Point Coaching. One nice thing about a group is that sometimes this offers the option...

...either as this also results in a generic invitation. 3) Connect with people you have really talked to The...include a note about where you met IRL (in real life) and perhaps what you talked about (or how good the... this rule, but not that many). I’m asked a lot about requesting connections to complete strangers for...have been able to tell when some start-ups were about to be bought by seeing certain groups of people ...using social media as a scientists Read blog posts about networking ...

...against the antigen bound to the plate. Figure 3: Overview of using Direct ELISA to quantitatively ...blog Learn about the advantages and disadvantages of using monoclonal antibodies. Read about other techniques... require two different antibodies. As mentioned above, it can also be used to characterize antibodies ...concentration of proteins in an ELISA well As mentioned above, the measurement of the concentration of proteins...

...make up the disulfide bonds between antibody chains. 3) Endogenous carbohydrates added to antibodies post-translationally...Check out our post on Biotinylation to learn more about biotin and its use in molecular biology. Other Conjugates...perfectly blissful, productive lives without thinking about these chemical reactions. After all, there is an...ready to use off the shelf. So why are we talking about this? Even though there are so many commercially...

...endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016/j.cell.2015.09.038 Harrington... is smaller than the traditional SpCas9 enzyme (about a third of the size) and does not require a tracrRNA...Cas12a to further optimize it for therapeutic and laboratory usage. Fast facts – PAM requirement: TTN or ... the lack of collateral cleavage). You can read about Cas7-11 in our blog post on its development and ...

...PMID: 24529477. PubMed Central PMCID: PMC4139937. 3. Anders, Carolin, et al. "Structural basis of PAM-...With this new found structural knowledge, in collaboration with the Joung Lab, they used targeted mutagenesis...edits in mammalian cells and may decrease worries about off target effects in applied and/or therapeutic...at Addgene. Resources on the Addgene Blog Read about Cas9 Homolog Cpf1 Read How Non-Homologous End Joining...

... The McMurray lab found that low concentrations (≤3 mM) of GdnHCl can functionally rescue multiple Arg-mutant...glycosylation (CDG) are a group of rare inherited metabolic diseases with severe and life-threatening consequences...understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to immortalize CDG...discovery led them to investigate the use of a common laboratory chemical, guanidine hydrochloride, as a potential...

...one locus (e.g. Cre) and heterozygous at the other. 3. Breed your F1 generation Now, your goal is to generate...homozygous alleles at both of your genes of interest. About 1 in 4 mice will be homozygous for the mutation ...Find our Plasmids 101 blog posts Read blog posts about using Cre-lox Read our genome engineering blog posts... posts Resources on Addgene.org Find more about Cre-lox Read our Genome Engineering Guide Learn more...

...Genetic Analyses in Mice. 2004. PubMed PMID: 14723844. 3. Atasoy D, Aponte Y, Su HH, and Sternson SM. 2008.... Blog  Read about Other Means of Genome Engineering Using FLEx AAV Vectors? Learn about Viral Vector ...12665802. 2. Branda, CS and Dymecki, SM. Talking about a Revolution: The Impact of Site-Specific Recombinases...

...PMID: 16836756. PubMed Central PMCID: PMC1534021. 3. Zhang, Bing, et al. "The significance of controlled...drawback of this approach is that it can be quite labor intensive. Target cells are transduced with serial...

... 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller...a 24-well cell culture treated plate. Seed 5 x 10 3 HeLa cells per well. Allow the HeLa cells to grow ... min in 500 µL PBS on a rocking platform. Section 3: Labeling with antibody Block for 20 min at RT on ...water Microscope slide Anti-fade mounting medium Laboratory wipes 15 mL conical tubes 50 mL conical tubes...paraformaldehyde and follow your institution's laboratory safety guidelines for disposing of waste in the...remove the coverslip. Blot the coverslip with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade...

... make it easy for your supervisor to support you. 3) Explain the problem and leave your emotions out of... I heard about supervisors in all of the above categories. In addition, I also learned about two other...Complainers, who spend most of their time moaning about their troubles, Silent or unresponsive clams, ... students and postdoctoral fellows are hesitant about expressing their ideas, especially if their supervisors...it is interfering with your work. Avoid talking about your emotions, such as frustration or anger, because... like the problem to be resolved You know more about your work than your supervisor does. You can save...Listen to your supervisor’s viewpoint and brainstorm about mutually beneficial solutions Your supervisor might...

...0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...interest Triton X-100 Benzonase/DNAse I (Millipore 71205-3) 40% Polyethylene Glycol 8000 (PEG) + 0.5 M NaCl Cell... PBS and add 35 mL of 0.05% Trypsin/EDTA. Wait ~2-3 minutes for cells to detach. Gently tap the sides ...stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow full precipitation. Precipitation... at 4 °C if needed. Transfer the entire sample to 3 x 500 mL conical bottles and centrifuge at 3900 rpm....8-fold. See the recipe for D1 + 0.1 M sorbitol above. Carefully pour off the media into a waste container...in a total of 5 mL of cell lysis buffer (recipe above). Pipet back and forth to resuspend each pellet ...

...two 96 well plates in just under 3 hours or one 96 well plate in about an hour. Automating vial fiIling... work into finding robots that’ll take a little labor off our hands. Good news! There is probably a machine...96 transformations in one run compared to doing about 50 transformations in the same amount of time by...machinery we have been able to transform 96 samples in about 4 hours with a greater than 95% success rate! Automating...35 minutes compared to a lab assisstant filling about 200 vials in 45 minutes by hand. How can you start...on each tube by hand. We’ll be sure to tell you about more lab updates as we develop them. In the meantime...

...Originally published Aug 3, 2017 and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017...ask the depositing laboratory to review the discrepancies. If the depositing laboratory confirms that these...finish the library preparation process only takes about one day, even for six plates (576 individual plasmids... we perform a 2x251 run on our MiSeq that takes about two days to complete. After the run completes, we..." In some cases, there will be more information about our sequencing results in the Depositor Comments...to note the appropriate barcode for each sample. About 24 hours later we unload the FASTQ data from our...

...directly back to your specific goal. When thinking about step 3, however, you may be surprised at the number...Delivery methods for mammalian cell lines As mentioned above, the expression system you choose in many ways dictates.... The user should review the current literature about their preferred model.  If your expression system... Check out our CRISPR Featured Topic Page Learn about CRISPR Delivery Using Ribonucleoproteins (RNPs) ...

...deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team will...streptomycin, tetracycline, combinations of the above, or other. High or Low Copy Choose from: high...the “Purpose” field. If you ever have a question about what should go into a cell, hover over the cell ...Addgene and contact your technology transfer office about the MTA. Pro Tip! Download a new spreadsheet each...

...expression and high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type...Addgene blog Read more about using AAV for retinal gene therapy Find more about precise neuronal targeting...injected into macaques and postmortem human retinas. About 25% of these AAV synthetic promoters successfully...

...more advantageous for your search. Timesaving Tip #3: Note that protein databases available are unlikely...such as full plasmid sequences provided by the laboratories that deposit their plasmids with us or other...mismatches and gaps in the alignment. If you are curious about the differences in the blastn programs, check out...Inside Look at NGS Plasmid Quality Control Learn about our Snapgene-powered plasmid maps. Resources on...

...PMID: 24552514. PubMed Central PMCID: PMC3929550. 3. Kaati, Gunnar, Lars O. Bygren, and Soren Edvinsson... the histone protein of interest (in the figure above, H3 at K9 or K27). Joined to this by a flexible ... in Your Experiments Post Read Other Blog Posts about Fluorescent Proteins  ...

... 1 µg DNA 1 µL of each Restriction Enzyme 3 µL 10x Buffer 3 µL 10x BSA (if recommended) x µL dH 2 O (to... T4 DNA Polymerase or Klenow DNA Polymerase I for 3′ overhang removal and 5′ overhang fill-in. If you ...more at (Link opens in a new window) NEB's website about star activity . If you are digesting a large number...

...end that is identical to an adjacent segment and a 3′ end that anneals to the target sequence. One strategy...reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary...) to the isothermal reaction mix. ET SSB protects 3’ ssDNA ends from the ssDNA-specific endonuclease activity... the right). When designing your plasmid, think about what DNA segments you will need to join to create...

...of amino acids. Each amino acid is encoded for by 3 nucleotides termed a codon. As there are only 20 natural.... Uracil replaces thymine in RNA molecules. Every 3 nucleotides (codons) in a DNA sequence encodes for...Reference Page Plasmids 101: How to Name Your Plasmid in 3 Easy Steps Molecular Cloning Techniques Reference ...human genome with restriction enzymes would yield about two million DNA fragments, which is far too many...the molecular biologist’s toolbox. To learn more about different types of cloning methods check out our...restriction enzyme digestion. For more information about restriction enzymes check out NEB's website . Insert...endogenous expression levels). For more information about promoters, both bacterial and eukaryotic, as well...

...Cells Day 2–3 (am): Remove media, replace with fresh media containing selection reagent Day 3–14: Change...300 200 1:10 150 350 1:50 30 470 1:100 15 485 1:500 3 497 Add 0.5 mL of a single viral dilution to each ...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...cells in the untransduced well (0 µL lentivirus, above) are dying. Perform regular media changes and monitor...

...PMID: 25357182. PubMed Central PMCID: PMC4289409. 3. Rouet, Romain, et al. "Receptor-Mediated Delivery...scientists to make genomic alterations, bringing about a revolution in genome engineering, with new techniques...available by both the Jacob Corn and Alex Schier laboratories. Delivering Cas9-gRNA ribonucleoproteins  An...delivery of RNPs is electroporation (A in the figure above), which generates pores in the cell membrane, allowing...as lipid-mediated transfection (B in the figure above), are in the early stages of development. David ...proteins harboring receptor ligands (C in the figure above), which result in the internalization of Cas9-gRNA...show promise for in vivo studies, little is known about the immunogenicity and stability of the Cas9 protein...

...important is letting people know it’s happening. Aim for 3-4 weeks advance notice for the first meeting so that...think about. Choosing a topic and gauging interest The first thing to do if you’ve thought about starting... discussions. Beyond the excitement of learning about new cutting edge science, researchers of all varieties...help stay current on new and emerging work by collaboratively discussing recent publications in a specific...up. At Addgene, we wanted to think more closely about the wealth of high-throughput sequencing (HTS) data...the paper.  Before the first journal club, think about the meeting itself A journal club should aim to ...published science. They also serve as a way to learn about work outside of one's own field in a structured ...

...Amplifying genetic logic gates." Science. 340, 599-603 (3 May 2013). Vivek Mutalik et al. "Precise and reliable...Foundation's Linda Kahl explains the Addgene collaboration this way: "Addgene is one of the leaders in ... our core values." Addgene spoke with Endy more about the BIOFAB and BIL gates plasmid kits, the Biobricks...reference to George Boole who in 1854 was thinking about logic and analyzing human language and observed ... freely available? Endy: When we start thinking about languages, humans have two types of languages: one...does; it's free to use and that tends to be true about most successful human-to-human languages. Who owns...signals. Let's say I wanted to detect every primary metabolite in a cell. The only way to do that at scale right...

... of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing world of genome...have been written about CRISPR alone, and Zimmer himself has written entire books about optogenetics. Defining... been thinking about a long time. For the scientists who like to learn everything about one specific topic...wisely makes no such attempt. He is transparent about his own biases--many of which will be shared by ... a massive, expensive project that required collaboration from scientists around the world. The paper ...As a scientist who enjoys learning a little bit about many different things (part of the reason Addgene...

...the IPD of a cation channel, such as the serotonin 3 receptor, will promote depolarization in neurons. ...posts Read blog posts about neuroscience Resources on addgene.org Learn more about chemogenetics in our...as a DREADD agonist due to its ability to be metabolized into clozapine (CLZ). While some scientists argue...

...2013 Mar 19;110(12):E1152-61 Pubmed PMID 23487772. 3. Etessami R, Conzelmann KK, Fadai-Ghotbi B, Natelson...connected to the neck. Shortly after, we learned about organs and the jobs they perform. This became foundational...part of the answer to many biological questions about the brain. To address this, molecular tools to map...

...quantitative real-time PCR. J Microbiol Methods. 2006 Jun;65(3):476-87. doi: 10.1016/j.mimet.2005.09.007. Epub 2005...controls must be run in triplicate.  The nice thing about qPCR is that since it measures relative quantification...number, albeit within a range of RQ values as noted above.   In both applications, results can be affected...

...recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the end...Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested first thing in ...µL micropipette tips in a -20 °C freezer. Aliquot 3 mL SOC into each of four 14 mL Vented Falcon Tubes...cuvette and add to 14 mL vented Falcon Tube containing 3 mL SOC. Repeat for each of the 25 µl aliquots of cell...Vented Falcon Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate...require modifications dictated by the originating laboratory for optimal results. If you obtained the pooled...

...Bachle and Ina Ersing Seeding Labs and Addgene collaborate to provide scientific resources, equipment, and...discovery, and synthetic biology. Read on to learn more about the five awardees and their promising research projects... to the Addgene blog for updates and future collaborations. Many thanks to all of the awardees, all who...Additional Resources on the Addgene Blog Learn More About the Addgene-Seeding Labs Plasmid Grant Check Out...Expression Systems Synthetic Biology Resources Learn about Global Plasmid Distribution ...