...bands at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled.... Mol Biotechnol. 2008 Nov;40(3):252-60. doi: 10.1007/s12033-008-9082-3. Epub 2008 Jul 8. PMID: 18607781...plasmid sample. Troubleshooting Tips   Figure 3. Summary of plasmid multimerization troubleshooting...9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: 797459. Berg CM, Liu L, Coon M, Strausbaugh...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...expected fragments for that plasmid. This phenomenon indicates the presence of a multimer.   Fig. 1:...

...Seeger lab designed 3 different mRNA sybody libraries for use in the protocol, which cover 3 naturally-occurring...biotinylated (for purification purposes) protein target of interest, the protocol goes through 3 major steps... against membrane protein targets in as little as 3 weeks (Zimmermann et al., 2020). This selection platform...structures (concave, loop, and convex). Each of these 3 mRNA libraries have a concave, loop, or convex scaffold...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter Nanobody Fusions Plasmids...

...shRNA cassettes into the unique SnaBI site in the 3´-LTR). See here for other versions of pULTRA. Moore...included to monitor expression Parijs 11619 pLB 3rd Modification of pLL3.7; Genetic elements known to prevent...

... as their orientation relative to the gRNA (5’ or 3’). In some cases, there is no requirement, which is... spCas9 isn’t always the winner!).   Fig. 3 – Comparison of different Cas enzymes using CasPEDIA...established uses, and intended applications. Within CasPEDIA, there is a classification system, CasID, to help...multiplex ability Experimental guidelines and applications Protein structure and properties Links to ...you want to read literature on the less common applications of an enzyme for your non-traditional experiment...al. (2023). CasPEDIA Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint...I., Iranzo, J., et al. (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and...

... of the start of the gene and a HindIII site just 3’ of the end of the gene. This way you can then cut...DNA as this will be useful for the ligation step. 3. Ligate Your Insert into Your Vector In the ligation...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies, it...equivalent numbers of colonies on each plate (unsuccessful 3).     If you have a high number of colonies on your...equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid...colonies and check them for successful ligation. Pick 3-10 colonies depending on the number of background ...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...

...sequences at its 3’ and 5’ ends that are crucial cis-acting signals for genome replication and viral gene...genome. It contains the 3’ and 5’ ends of the genome which are required for replication and transcription (...synthesis at the 3’ end of the minigenome to produce the positive sense, genome replicative intermediate ... PMID:15298166. 3. Hoenen, Thomas, et al. "Minigenomes, transcription and replication competent virus-...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola virus...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...

...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...

... and breathing, and often results in death within 3-5 years from symptom onset. Disease mechanisms are...loci seem to be associated with the disease and implicate a wide range of cellular processes. The vast majority... consists mainly of managing symptoms through medication and surgery. PD primarily involves the malfunction...available from Addgene, sortable by type of genetic modification and target species. You can also find our guide...Sun et al. Nat Methods. 2020 Oct 21. Glutamate indicators with improved activation kinetics and localization...A multicolor atlas for whole-brain neuronal identification in C. elegans. Yemini et al. Cell. 2021 Jan...Alzforum is a news website and information resource dedicated to helping researchers accelerate discovery and...

...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...

...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! ...

... 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 ...

...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...

...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...

...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...

...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...

...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...