...two regions: the 3’ end binds template DNA for PCR amplification, while modifications and homologous sequences... the whole vector sequence between modification sites (see Figure 3), producing multiple fragments with... while adding a GFP-tag (Figure 3). Achieving multiple modifications requires multiple recombination ...not easy to amplify by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not...PubMed PMID: 8346047. PubMed Central PMCID: PMC331480. 3. García-Nafría, Javier, Jake F. Watson, and Ingo H...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...’ Check your amplification by gel electrophoresis. In our experience, good amplification after PCR means...

...desired gRNA target sequence, as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... proteins in a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple...genes in human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of...to specify the reading frame of the insertion, and 3) a vector containing the desired tag, which can be...Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.” Genetics. 202(4)...

... (i.e. DsRed) [3]. This is a problem for fusion proteins. One of the major applications of FPs is to visualize...potential to form large stable polymers of dimers. 3) FRET biosensors that contain two dimerizing FPs can...localize incorrectly and may function improperly. 3. Incorporation into a FRET biosensor A FRET biosensor...DsRed or high affinity FPs such as TagRFP. Problem 3 can be solved, in most cases, by using monomeric FPs...2002. 296(5569): p. 913-6. PubMed PMID: 11988576. 3. Matz, M.V., et al., Fluorescent proteins from nonbioluminescent...even if they were described as monomeric in a publication. The gold standard in assays used to determine...An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein...

...manipulate, 2) they have long-term expression; and 3) they have limited toxicity. However, a key challenge...1x1011 vs 1x1012 viral genomes (vg) per mouse. Figure 3 shows a gross comparison of AAV.PHP.eB and AAV.PHP.S...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 2e in Chan...transduction, measured via GFP expression, was determined 3 weeks later. Table was derived from figure 3b in Chan...PubMed PMID: 26829320. PubMed Central PMCID: 5088052. 3. Lentz TB, Gray SJ, Samulski RJ. Viral Vectors for... creates a template that’s suitable for PCR amplification using pre-designed primers. By using this library...morphologic studies), as well as translational applications (mechanistic studies and gene therapy treatments...

... improving cross team communication has helped Addgene move from completing 2-3 large projects a year,...way is not necessarily easy, so we came up with a 3-part strategy: Train: Nearly every new Addgene employee.... I needed to a) figure out the sources of miscommunication and b) find practical solutions.  My PhD ... for this task. Good scientists learn how to communicate their work differently based on their audience... family is going to vary widely. The way you communicate your science at a poster is going to vary depending...two main problems with Addgene’s cross team communication. Although these real world examples did not ... for the container to be used in cross-team communication A list of “AKA” names, or jargon terms, for ...

...may be wondering, “why is it necessary to have all 3 of the different binary systems above if they all ... or look at multiple genes at the same time.  The 3 binary systems listed above are orthogonal, meaning... synthetic systems. Recent work has even used all 3 in a single modular vector for ease of use! Conclusion...313. doi: 10.1007/s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122. Pfeiffer, Barret D., et al. “Refinement..., lineage tracing, and mosaic analysis.” Cell 141(3) (2010): 536-48. PubMed PMID: 20434990. PubMed Central...reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed PMID: 25581800. PubMed Central... also been developed which don’t fit in either category, but those are a subject for a future post! ...

...usually 1-3 of the first 3 bps are modified) for optimal stability and efficacy.  Base modifications What ... in the sugar ring, a popular stabilizing modification is 3’ phosphorothioate linkage, where a sulfur ...crRNA also contains a ~20 bp repeat region on its 3’ end. This repeat region associates with the trans-activating...switches. Stabilizing gRNA modifications  Sugar phosphate backbone modifications The recognition sequence...phosphorothioates with 2’-O-methyl modifications, proved more stability than just one modification alone (Hendel, et al...ordered as synthesis modifications when purchasing your gRNA. Pro tip: modifications are typically incorporated...stabilizing modifications listed above were developed before CRISPR-Cas9, for use in applications like siRNA...

... break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a substrate for... repair (DSBR) In the classical DSBR pathway, the 3’ ends invade an intact homologous template to serve...the non-invading strand at the other DSB end. The 3’ end of the non-invasive strand is elongated and ligated... donor DNA. Richardson CD, et al. Nat Biotech. 34(3); (2016). PubMed. Efficient homology-directed repair...Non-Homologous End Joining Learn about Base Editing Read 3 Tips to Improve HDR Efficiency for CRISPR Editing ...pathways of DSBs, with a focus on repair for genome modification purposes. How does homology directed repair...specific mutations into the damaged DNA if these modifications exist in the template DNA. There are several...

...Scientific reports 3 (2013). PubMed PMID: 24284873. PubMed Central PMCID: PMC3842082. 3. Mashiko, Daisuke... It can take roughly 3 months from the day you decide to do a gene modification experiment in mice to...pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter....classified into four groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control...control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...effective ones to increase your chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs...

...antigen. J Gen Virol., 73 (Pt 3), 653–660. https://doi.org/10.1099/0022-1317-73-3-653. PMID 1372038. Randall...comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer signals due to ...autofluorescent tissues like the intestine. Figure 3: Expression of fluorophore-tagged GOLG-4 through eight...disruption in Mycobacterium abscessus. J Bacteriol., 206(3), e0033523. https://doi.org/10.1128/jb.00335-23.  ...performs well in many different antibody-based applications.  IPI’s recombinant version is a fusion of mouse...with additional cofactors or using significantly truncated forms that limit its functionality. To make biophysical...cellular processes in C. elegans: mScarlet3 for applications needing intense brightness (such as low-expression...

...Recombinant DNA by CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F ...Microbiol. 1995 Mar;15(6):1031-7. PubMed PMID: 7623659. 3. Bernard, P., et al. “Positive-selection vectors using...DNA cleavage by gyrase.” J Mol Biol. 1993 Dec 5;234(3):534-41. PubMed PMID: 8254658. 5. Bernard, P. and ...DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed PMID: 1324324. 6. Tam, JE. and Kline...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful...

...." F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central PMCID: PMC4304227. 3. Sauermann, Henry...positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway into academia..., Eve. "Looking out for future scientists." eLife 3 (2014): e04901. Pubmed PMID: 25291257. Pubmed Central...University of Massachusetts Medical School. Graduate education programs are also beginning to address these issues...The panel discussion on Friday morning will be dedicated to Academic data and the Labor Market. On this...Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic Medicine...disease.  Outside the lab, he is interested in communicating science and in the intersection between science...

...three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering with...DNA introduced into the E. coli. Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease.  Exo will degrade linear... 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough, a ...the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview of ds-... integrated defective prophage 2) from a plasmid, 3) from mini-λ or 4) from the lambda red phage itself...PubMed PMID: 10811905.PubMed Central PMCID: PMC18544. 3. Murphy, K. 2016. λ Recombination and Recombineering...of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites...

...crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA:... Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary culture of mouse neurons and ...and GFP.  Of these neurons, ~15% had indels at all 3 targeted loci.  See the chart below for a summary ...Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227. PubMed Central PMCID... a simple and effective method with multiple applications (in vitro, in vivo, transfection, viral transduction...Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background with Our Guide Pages...

...where the lab magic is happening. 3) Volunteer – Step into science education Do you aim to reach young people...-to-day business with popular work experiences of 3-10 days. And the Society of Experimental Biology offers...science communication are as varied as the field itself. Among other things, science communication comprises...certain aspects of art and education. If you suspect that science communication might be for you, don’t ... networking with your local education community. Get involved in education advocacy non-profits like Phipps... Training – Get a formal education Some people get into science communication by taking a media or journalism...project. Science communication opens doors Getting involved in science communication has been the most...

...PMID: 27175853. Pubmed Central PMCID: PMC4866822. 3. McDowell, Gary S., et al. "Shaping the Future of ...perspective from junior scientists."F1000Research 3 (2014). Pubmed PMID: 25653845. Pubmed Central PMCID...Riglar Advocating for Science Symposium and Workshop 2016 – Sept 16-17 MIT, Boston The Advocating for Science...futureofresearch.org/advocating-for-science-boston-2016/. Sign Up For the Advocating for Science Symposium...Director of Media Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner...union dues. Time will tell the extent of the implications of this ruling and whether graduate students...labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy of...

...field, (2) that you have three 3 years of research experience in the field, (3) that you have an offer of ...employment based green card applications and approved 91,023 during the first 3 quarters of 2017. As an international...job offer is needed for this category. To qualify, you need to satisfy 3 out of the 10 criteria listed...well positioned to advance the proposed endeavor; (3) The foreign national’s proposed endeavor will be ...institute. For the EB-1B, you need to prove 3 threshold requirements: (1) that you have international ... you must show that your company employs at least 3 full-time people engaged in researcher activities....higher education nonprofit entities related to or affiliated with institutions of higher education nonprofit...

...MSOE center for BioMolecular Modeling and the CEO of 3-D Molecular Designs. Have you ever held your favorite...models were useful in that they not only traced the 3-D path of the protein’s backbone in three-dimensional...arginine 53 really is right there, at the end of helix 3…just like we said it was”.   Seeing is believing. ...MSOE center for BioMolecular Modeling and the CEO of 3-D Molecular Designs.     Additional Resources on the...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...biologists have always struggled to effectively communicate the nuances of protein structure in a flat 2-...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...

...plasmids ready for deep mutational scanning.     3 reasons to be interested in deep mutational scanning...polymerase, 2) increased PCR extension time, 3) increased concentration of MgCl2, 4) increased concentration ...synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is degraded...mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site or multi-site saturation...Mutagenesis Protocols, pg. 103-109. PubMed PMID: 20676978. 3. Wrenbeck, Emily E., Justin R. Klesmith, James A. ...created by making some or all of the following modifications: 1) increased concentration of Taq polymerase...

... seconds for 3 hours. Image from Lee et al., 2020. SPOTlight was used to image over 3 million cell...investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons. Casɸ ...For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or C). The authors demonstrate ...including excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases...BioRender.com In their recent ACS Synthetic Biology publication, Mary Dunlop’s lab generated a photosensitive...regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd dimerizes bringing the C-terminal...