...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...

...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...

... 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...

...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...

... 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...

...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...

...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...

...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) ...

...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...

...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...