...do an AMA in the first place and give you some reasons why you should consider using Reddit to share your... as our first foray into Reddit. Here are some reasons why: 1. Reddit has a large and active community...

...tests. All sample comparisons Do you really need to know every pairwise comparison within your population...we will dive into the nitty gritty details of comparison statistical tests – when to use them, what they...one-tailed test also works for exclusively less-than comparisons. Paired and unpaired t-tests Samples that come...family-wise error rate by only performing the comparisons you really care about. Family-wise error is the...probability of a false positive occurring when many comparisons are performed. For example, if your alpha is ...you have 5 samples and perform every pairwise comparison (10 in total), your likelihood of a false positive...higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise error, is the false discovery...

...eukaryotic genomes.  Components of a transposon system Transposon: The transposon is the DNA sequence that moves...also called retrotransposons, and Class II TEs, also called DNA transposons. DNA transposons are commonly...discovered transposons due to changes in coloration of corn kernels. Aside from corn, transposons are in ...2010).  So why do transposons matter? The outcome really depends on where a transposon lands. For example...components of transposons, how they are classified, and how they are used in the lab. What is a transposon? DNA...Transposable elements (TEs): Retrotransposons  Class I TEs are also known as retrotransposons. They transpose by...which are required for LTR retrotransposon transposition. Non-LTR retrotransposons contain two open reading...

...research findings so difficult to do?  There are four reasons that research can be difficult to replicate. Unclear...with powerful cognitive biases such as motivated reasoning and hindsight bias, we may fool ourselves and ...Unanticipated discoveries Finally, there are good reasons for obtaining a different result after conducting...

...contact to connect on LinkedIn and always include a personal note about where you met and what you discussed...the near future. Even just one more meeting (in person or virtually) will make the relationship stronger... law of networking--don’t be creepy. There is a person on LinkedIn I once helped by answering one simple...not at all necessary. Once you’ve talked with a person a few times, if it was a positive experience for...was in graduate school, there was only one other person in my class who had children. When I had my baby...answered my questions and honestly, she was the only person who really understood what I was going through....

...conversations on the history of the group, lessons learned, and to Sonali Roy, Magdalena Julkowska, and Jennifer... provide greater visibility of these materials, Sonali Roy, a postdoctoral researcher at the Noble Research...effort. The future of reproducibility workshops and lessons learned: Since early 2018 the team has grown to...researchers at all levels of the scientific community. Lessons learned Instead of continuously talking about...

...Zhang group. Mechanism of CRISPR-transposons One of the CRISPR-transposon systems developed by the Sternberg...aka mini-transposon), which contains the DNA cargo of interest flanked by ~50-150 bp transposon end sequences...characteristic of the Tn7-family of transposons. Insertion of the mini-transposon can occur in two different orientations...with the same transposon exhibit reduced efficiencies (Vo et al., 2020). CRISPR-transposons within the CRISPR...Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic elements capable of spreading... Beauty). Despite their diverse utility, DNA transposons have been hindered by their limited programmability...recently surpassed with the discovery of CRISPR-transposons by the Sternberg and Zhang labs (Peters et al...

...the next steps. Harvard PI Angela DePace has put personal development at the center of her lab (pictured...easily adaptable for use in your lab! Promoting personal development through thoughtful planning and honest... of in the supplement of this paper), help each person organize his or her thoughts to facilitate honest...through obstacles, and take steps to resolve interpersonal conflicts in the lab. Yearly planning meetings...hear about the steps taken in your lab to promote personal development - let us know your strategies in the... personal development...

... said Addgene’s Lianna Swanson, who has been working with members of Davidson's lab to coordinate the ...work of Michael Davidson and his colleagues." Acknowledgments Thank you to Michael Davidson, Michelle Baird...and their use for imaging cell biology, Michael Davidson’s lab at Florida State University has been the ... easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection webpage. These...who has created plasmids as a technician in the Davidson lab for the last four years. “Researchers can ...comprehensive list of readily available targets since the Davidson lab always tagged new proteins with mEmerald first...to localize to specific cellular structures.The Davidson lab even included some of their own reference ...

...the basics of Gibson assembly as a parody of “Breakfast at Tiffany’s.” Overview of Gibson assembly The ...The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. Craig Venter...program to design primers for Gibson Assembly. For a simple example of using Gibson assembly, imagine that you...the mixing and matching of parts. Gibson assembly meets CRISPR Gibson can be adapted to more complicated... a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic... have become more and more common, and for good reason. They offer many advantages over the traditional...disadvantages, and examples of how scientists are using Gibson assembly to put together DNA fragments. As a fun...

...announce the recipients of this year’s Michael Davidson and Roger Tsien Commemorative Conference Awards...of their choice while honoring the late Michael Davidson and Roger Tsien who have played enormous roles...made some changes. In addition to supporting in-person conference attendance, the award now supports virtual... Aggarwal, and colleagues Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo...

...Housden BE, Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods...976 . https://doi.org/10.1038/nmeth.2600 Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP...

...but also a biological transposon system. Sleeping Beauty is a synthetic transposon system that can be used...resurrecting this transposon system? Scientists can use Sleeping Beauty transposons as a non-viral system...Now, we have meetings in rooms such as Groucho, Sonic Hedgehog, Sleeping Beauty, Cookie Monster, Spaghetti...Shivdasani Lab from the Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development...The largest conference room at Addgene is named Sonic Hedgehog but this is not a direct reference to the...gene in animal development. To understand where sonic hedgehog got its name, we need to go back to 1980...Wiechaus, 1980). The mammalian hedgehog protein, sonic hedgehog (SHH) was named by a postdoc in Cliff Tabin...

...My son is an engineer and my daughter has a business degree. From the very start of their undergraduate...

...family, he did not seem much different than my second son of similar age. When he came to visit us and watch...  Many thanks to our guest blogger Kurt Swanson! Kurt Swanson is a structural biologist and protein engineer...This post was contributed by Kurt Swanson a structural biologist and protein engineer currently working...

...Current Protocols in Protein Science. John Wiley & Sons, Inc. https://doi.org/10.1002/0471140864 Fox, J....

... a mCherry OptoNB to the catalytic domain of the Sons of Sevenless (SOS), a guanine nucleotide exchange...

...Transposagen What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths...until 2005 [1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The ...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater number...ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source ...you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences distributed...genome. We encourage you to deposit your piggyBac™ transposon vectors with us to help us expand this useful...transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed...

...important for social and regulatory reasons. 1.) The transposon consists of a mirrored set of inverted....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons...the site of insertion. In both of these cases, transposons combine the advantages of viruses and naked DNA...mechanisms. The use of non-viral vectors, like transposons, avoids many, though not all, of these defenses...reaction occurs which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide...plasmid is empty following the removal of the transposon; the plasmid is then degraded by the cell.4.)...

...but he or she probably has reasons for it. Do not take criticism personally or get defensive. Look at ...important issues in person or over the telephone instead of email. As you cannot see a person’s body language...get nods from nearly every participant. Robert Bramson, author “Coping with Difficult People,” identified...specific strategies to deal with each of these personality types is beyond the scope of this article, there...resolve conflicts What Is Assertiveness? An assertive person is able to communicate their ideas confidently ...your supervisor, even if he or she is a difficult person: 1) Understand your supervisor’s expectations ...or she would like a written report or to meet in person.   Regardless of your supervisor’s management style...

...were usually the products of teams: Watson, Crick, & Franklin, Meselson-Stahl, Hershey-Chase...Who was I ...intelligent, motivated person really could know a little bit about everything or in Jefferson’s case, even a lot...work of more than one person. A selfish part of you might not want another person’s name on your next paper...clear why that person is there. If it becomes clear after a few meetings that a person is neither adding...all our externally facing kit pages. No single person could possibly understand every nuance of how kits...should have a goal, or at the very least, a clear reason for existing. Here are a few types of teams that... - with the possible exception of when Thomas Jefferson dined alone.” There was a time in human history...

...to the conclusion that a conflict is personal (”Person A and Person B just don’t get along”) when in fact...conflict are substantive (“Person C told Person A one thing and inadvertently told Person B something else.")...also be categorized as either “personal” or “substantive”. A personality-based conflict occurs when two...multiple channels is not reasonable or sustainable. That said, when you know that a person or team should be ...large organization, because confirming that every person has read and understood every communication that...assign the responsibility in question to a specific person or team. What Makes You a Good Conflict Resolver... for a fight. But you also can’t be the type of person who avoids conflict at all costs or actively runs...

... result of the necessary restrictions on close personal contacts. However, that should not deter any student... letter can be found on the Addgene blog.   3. Personal statement brilliance In addition to sending in... cover letter, you are always asked to draft a personal statement, which is the perfect opportunity to...questions, thus devoting some time to writing a solid personal statement will pay dividends. This is the only...and forward-looking. Embrace it, and let your personality leap out of the page. Finally, as previously ...find the tools that can help you craft a solid personal statement.  4. Letters of recommendation wisdom...to vouch for the character you painted in your personal statement. Make certain to choose your recommenders...

... year’s Michael Davidson and Roger Tsien Commemorative Travel Awards: Melissa Chiasson, Elizabeth Unger...variation at large scales and making personalized medicine a reality,” Chiasson explains. Elizabeth Unger: Developing...The late Michael Davidson and Roger Tsien played enormous roles in fluorescent protein development by...To honor their legacy, we launched the Michael Davidson and Roger Tsien Commemorative Travel Awards in...Unger, and Michal Bernach. Melissa Chiasson: Quantifying the abundance of thousands of protein variants ...time is time consuming and unscalable.  Melissa Chiasson, a graduate student in Douglas Fowler's lab at...from each bin are then extracted and sequenced. Chiasson is currently using VAMP-seq to study vitamin K...

... A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing... Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu,.... coli Ec48 retron RT and the S. pombe Tf1 retrotransposon RT, respectively. Their small size comes at... even in contexts where expression was reduced (Nelson et al., 2022). PE7: An alternate approach to RNA...RNA stability This recent contribution by the Adamson lab also addresses the problem of RNA stability....References Anzalone, A. V., Gao, X. D., Podracky, C. J., Nelson, A. T., Koblan, L. W., Raguram, A., Levy, J. M....A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019...

... A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024). Improving prime editing... Chen, C., Nelson, J. W., Newby, G. A., Sahin, M., Osborn, M. J., Weissman, J. S., Adamson, B., & Liu,...-RNA base pairing with the primer binding site (Nelson et al., 2022). PE7: This editor includes the fused...A. A., Koblan, L. W., Levy, J. M., Chen, P. J., Wilson, C., Newby, G. A., Raguram, A., & Liu, D. R. (2019...e29. https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette,...Ravisankar, P., Ward, C. C., Levesque, S., Jing, Y., Simpson, D., Zhao, A., Li, H., Yan, W., Goudy, L., Schmidt...

..., based on resorufin). SNAP-tag: In 2003, Kai Johnsson’s lab developed a self-labeling fusion tag based...Addgene’s collection. CLIP-tag: Also in 2008, Kai Johnsson’s lab reported another AGT-based tag named CLIP-tag...STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal microscopy. Image adapted...159 aa) are smaller than HaloTag (295 aa) – for comparison, common fluorescent proteins are typically 230...Wissner, R. F., Xi, Z., Allgeyer, E. S., Zhong, S., Thompson, A. D., Lowe, N., Butler, R., Bewersdorf, J., ...Strategies Matter for Super-Resolution Microscopy: A Comparison between HaloTags and SNAP-tags. Cell Chemical...Corrêa, I. R., Kindermann, M., Beaufils, F., & Johnsson, K. (2008). An Engineered Protein Tag for Multiprotein...

...new variant was named ChrimsonR. Plasmids containing Chronos, Chrimson, ChrimsonR, CsChR, and CoChR have...inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al., Dev Cell...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...selecting the important elements from existing transposon and vector systems and creating an all-synthetic...British idiom for “taking by surprise”) by Margaret Robinson’s lab, and more recently used by Stephen Royle’...Shining a light on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes...include a pair of channelrhodopsins, Chronos and Chrimson, which have enabled the group to perform independent...

...everyone wins. What are the underlying reasons for this person’s request? Your colleague is if someone...need help from another person/team at the company. How do I negotiate with the person/team for help? What...can help you understand the perspective of all persons involved and be used as a guide to point you toward...important plans? What is most important to each person? Someone has to be the first author but another...agrees to pick up both children while the other person uses an available after-hours time slot.  You were...thoughtful about what is most important to each person. It can especially be difficult to think creatively...supervisors and lead to you being remembered as a person who tries to find solutions that benefit everyone...

...modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg and Zhang...pneumoniae. Robert Husson lab's M. tuberculosis CRISPRi plasmids A group from the Husson lab used CRISPRi...insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning. Streptomyces...Zhang labs developed CRISPR-transposons. The Sternberg lab's system called INTEGRATE (Insertion of Transposable...By using a multi-spacer CRISPR array, CRISPR-transposons can be multiplexed. Transcriptional repression...CRISPRi for Gram-negative and Gram-positive bacteria Jason Peters, Oren Rosenberg, and Carol Gross used two...https://doi.org/10.1038/s41564-018-0327-z Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP...

...compatible cohesive ends Engler et al., 2008 Gibson Cloning Gibson assembly method Single reaction, can assemble...Central PMCID: PMC2574415. 3. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO.Enzymatic assembly...terminus or the 3’ terminus of the ligation product. Comparison to other high throughput cloning strategies There... identical polyadenylation signals or promoters Gibson et al., 2009 MXS-Chaining, BioBricks, Bglbricks...Sladitschek and Neveu, 2015; Shetty et al., 2008; Anderson et al., 2010 Among the chaining-based methods...Additional Resources on the Addgene Blog Read about Gibson Assembly Learn About Other Plasmid Cloning Techniques... Find Plasmid Cloning Protocols References 1. Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin...

...process known as opsonization. In antibody-dependent complement deposition (ADCD), opsonization of IgG and ...isotype class or even subclass, and are part of the reason why different isotypes drive different types of... in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive when holes are punched...(ADCC) is a slightly more direct method than opsonization. In ADCC, IgG antibodies bind to a pathogen ..., our antibodies have induced “stabbing” and “poisoning,” but let’s be honest, the elimination style the...10.1038/74704. PMID: 10742152. Dunkelberger, J., Song, WC. Complement and its role in innate and adaptive...  Rachel Leeson...

... you want to make will happen.  There are many reasons to multiplex gRNAs, some being: Using dual nickases...the Bullock lab, and gRNAs can be inserted using Gibson Assembly or SLIC cloning methods. A BsaI-based ... up your multiplexed gRNAs with Golden Gate and Gibson assembly methods If you want to scale up the number...use some assembly methods such as Golden Gate or Gibson assembly. Golden Gate Assembly of gRNAs Golden ...supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including Standard PolIII-gRNA...Multiplexing in plants Qi-Jun Chen Lab Golden Gate/Gibson Assembly Multiplexing Plasmids: These plasmids ...you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs are inserted into the pCBC vectors...

...making the detection of rare events difficult. Dickinson et al. sought to create a new and efficient selection-based...Figure 1: Self-excising cassette schematic from Dickinson et al. (2015). Used with permission from the Genetics...your gene of interest into the FP-SEC vector using Gibson assembly. This construct will serve as the repair...for the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating the steps to fluorescently...Society of America. Applications of this system Dickinson et al. have developed a modular system that requires...with a Self-Excising Drug Selection Cassette. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein...

...background information. Your options for this step are sonication, which uses high-frequency sound waves to break...break up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher ...to the selective nature of the cutting. While sonication is usually the preferred method, enzymatic digestion...concentration.  Protein Capture After crosslinking and sonication or enzymatic digestion, you’re ready to incubate...availability, which could affect antibody selection. Sonication (or enzymatic digest) will also have to be optimized...fragment length for your output assay. A longer sonication time could mean needing to increase crosslinking...effects of increased sample agitation; a shorter sonication time could be an opportunity to decrease crosslinking...

...PubMed Central PMCID: PMC146043. 9. Nilsson, Stefan V., and Göran Magnusson. "Sealing of gaps in duplex DNA...gaps. For example, DNA assembly methods, such as Gibson Assembly® and NEBuilder® HiFi DNA Assembly, require...for a ligase? DNA ligases generally prefer fully Watson-Crick base-paired dsDNA substrates to those containing... base-pair size nearly indistinguishable from a Watson-Crick base pair, are joined with nearly the same...phosphate to the ligation). The structural/mechanistic reason for this differential is not known for certain,...allowed screening of all possible base combinations (Watson-Crick and mismatched) around the ligation junction....6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction...