...molecular targets under research include TIM-3, LAG-3, STAT-3, EGFR, CD20 and CD73 on specific solid as ...cell-surface antigen production to evade the immune system. 3. Escape - Tumor cells that survive this phase are ... situ before being injected back into the body in 3 doses intravenously. B cells and T cells activated...medicine 10.9 (2004): 942-949. PubMed PMID: 15322536. 3. Dunn, Gavin P., et al. "Cancer immunoediting: from...potential beneficiaries. Collaborative therapy For the most efficient outcomes, collaborative therapies must be... of the tolerance-producing antigens enunciated above. Finally, signals released from the microenvironment...are in their clinical trial phase. In all of the above therapeutics, techniques like genome engineering...

... 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 ... plasmids you need. If you'd ever like to write about a recent plasmid deposit please sign up here.  Here's...

... point for a new generation of ABEs), consists of 3 components:  a deaminase (TadA, originally from E.coli...al., 2020). The generated ABE8s needed to produce 3 concurrent base edits to withstand antibiotic selection...protospacer adjacent motif (PAM) made up of 5′-NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines...ABE8.20-m was the winner. It could simultaneously edit 3 genes (98.1%, 98.3%, or 98.6% efficiency, respectively...more blog posts about base editing Learn about another evolved base editor: ABE8e Read about xCas9, a CRISPR...

...Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required: intermediate Intramuscular Intramuscular...Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner Intradermal...Intraperitoneal Scruffing Peritoneal cavity 25–27 < 2–3 mL Intermediate Intramuscular Mechanical restrainer...Back or hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia...Routes of administration. In H. Hedrich (Ed.), The Laboratory Mouse. Academic Press. Turner, P. V., Brabb, ...M. A. (2011). Administration of substances to laboratory animals: routes of administration and factors...

...) 0:00-1:00 : Intro 1:01-3:48 : Kingdom Kwapata's background and education 3:49-4:35 : Kingdom on opportunities...more about Dr. Kwapata’s current work and thoughts on the need for more scientific collaboration across... connecting us with him. Learn more about our recent collaboration with Seeding Labs here. Special thanks...received an award from nonprofit and Addgene collaborator, Seeding Labs, for lab equipment that will help...Addgene Podcast.     Additional Resources Learn about Seeding Lab's Work with Lilongwe Univeristy of Agriculture...How You Can Contribute to the Addgene Blog Learn about Addgene Plasmid Donations for Rare Disease Research...

...plasmids requested each year by year of deposit (Figure 3), we saw that once CRISPR plasmids become popular,... used and available. If we look closely at Figure 3, which shows requested CRISPR plasmids categorized...requested and shipped out each year.     Figure 3: Percentage of requests per year for CRISPR plasmids...Figure 2, and the longevity of tools seen in Figure 3, indicates that the usefulness of popular CRISPR tools...with Addgene by year. (C) Cumulative number of laboratories that have deposited CRISPR plasmids by year....

... et al. created the 3rd generation system (Figure 3), which differs from the 2nd generation in two key...SIN) lentiviral vectors contain a deletion in the 3’ LTR that prevents aberrant activation of nearby genes...PubMed PMID: 9765382. PubMed Central PMCID: PMC110254. 3. In-vivo RGB marking and multicolour single-cell tracking...RA, Berinstein EM, Medin JA. Immunotherapy. 2015;7(3):271-84. PubMed PMID: 25804479. 9. Development of ...gene therapy applications. Read on to learn more about the current (and future) applications of lentiviral...do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1, well-known to...Libraries Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...

...contains two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able ...dsDNA cutting. A Dolcetto library containing only 3 sgRNAs (setA) also outperformed CRISPRi libraries ...PMID: 26780180. PubMed Central PMCID: PMC4744125.  3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated...Read this blog post about CRISPR Kinome Libraries available at Addgene Learn about the advantages of pooled...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources on...libraries for the mouse genome! Other Resources Read about CRISPRi and CRISPRa from the NIH E-newsletter ...

...CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target... the human genome, the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes...zygotes, the gRNA will seek out its target among the 3 X 109 nt of genetic content in the mouse genome and...predictable. In our best case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA locus as...lines. Second, the process is much quicker. It takes 3 months to generate founder mice using CRISPR, as compared... Delivery Methods Learn about Mouse Work on Exercise and Depression Read About Brainbow Resources on ...success is not always guaranteed. To learn more about this process check out our Mouse Modeling blog posts...

...name  Include information about the insert in your plasmid name. This is often a 3-6 letter representation...rat (rattus rattus or rattus norvegicus), etc. Step 3: Add your tags  Add any tags or fusions that are on... just by reading the name. For more information about plasmid features and tips you can use in the lab...

...as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed and nicked...The double digest with both HindIII and BamHI (lane 3) produces bands at 6kb and 1.2kb (red box), matching... and run on an agarose gel, you are likely to see 3 bands. This is due to the fact that the circular DNA...1kb ladder (lane 1) that has bands ranging from about 500bp to 10kb, with the 3.0kb fragment having increased...

...genome manipulation. Nature Reviews Genetics, 24(3), Article 3. https://doi.org/10.1038/s41576-022-00541-1 ...locations. This allowed us to train ePRIDICT (Figure 3), a model that predicts how well a genomic site can...influence editing results at your target. Figure 3: High-throughput screening of the effect of chromatin... al., 2023). We trained our models in close collaboration with the Krauthammer lab (University of Zurich...

...then binds the nicked DNA, and extends it from the 3’ end, while its native endonuclease activity degrades...dissociates and PolI binds (middle) and extends from the 3' end of the nick (right), occasionally introducing... which increased EvolvR’s mutagenesis rate to ~10-3 mutations per nucleotide per generation, and (2) the...blog Learn about other CRISPR methods for bacterial genome engineering   Learn more about lambda red ... genetic variants in bacteria. Read on to learn about the key components of EvolvR and its potential applications...

...podcast from producers Tyler Ford and Eddy Page. 2:00 - 3:55 : Alex introduces himself and explains why he decided...decided to do an MD PhD - it's all in the family. 3:56 - 7:04 : Alex describes the MD PhD process and ...Additional Resources on the Addgene Blog Learn More About Alex's Work with Cas9 Activators Michael Koeris ...Pages Find Ready-to-Use CRISPR Lentivirus Learn about Other Members of the Addgene Leadership Team ...

...abstracts about research: www.WeShareScience.org. WeShareScience is a free platform for sharing short, 3 to ...terms of communicating ideas. In 2012, for example, 3 billion hours of video were watched each month, in...means for communicating research. Short videos of 3 to 5 minutes provide more than enough time for researchers...by the numbers." Nature 525.7569 (2015): 306-307. 3. Spicer, Scott. "Exploring video abstracts in science...learn about how researchers are using “big data” to look at Lyme Disease in new ways. Or learn about the...ot­her disciplines, are even less likely to read about our work. We therefore must discover new ways to.... Only on occasion do we then have time to read about research from other disciplines, or from other countries...

...genetics. https://doi.org/10.1007/s00294-019-01040-3 3. Khatibi et al. 2021.  “CRISPR Genome Editing Technology...derived from iPSCs. From Rohn et al. 2018; see figure 3 for full details.    CRISPR advances in monoallelic...therapeutic.” There are many fundamental questions about CRISPR/Cas biology still left to answer as well,...

...Aggarwal et al…, Supplementary Figure 3.   Figure 3: pH titration curves and ∆F/F0 for the... kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for...Additional Resources on the Addgene Blog Learn about other Fluorescent Biosensors A Practical Approach...

...Part 2 here! Time : Topic 0:00 - 0:50 : Intro 0:51 - 3:08 : The importance of developing a strong graduate... need for belonging in the scientific environment 3:09 - 5:58 : Specific Examples where Niroshi and Ben...How did you hear about post grad opportunities and when did you start thinking about them? 18:30 - 19:... top if you leverage your community, think hard about picking a good mentor, and begin considering career...19:54 : How to think about picking a post-doc project 19:55 - 22:40 : Improvements that can be made to ...

... a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal...sequences for the commonly used S. pyogenes Cas9 (3'-NGG) are abundant throughout the human genome, they... derived from species other than S. pyogenes, and 3) the use of non-Cas9 enzymes. (For more details on...PAM sequence ’) Streptococcus pyogenes (Sp) 3' NGG Staphylococcus aureus (Sa) NGRRT or NGRRN...Resources on the Addgene Blog  Read more about Cpf1 Read more about SaCas9 and AAV Check out our CRISPR Featured... and VRER) recognize novel PAM sequences (shown above). The VQR, EQR, and VRER Cas9 variants are capable...example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...

...optimizing “cut-to-mutation distance”. Furthermore (3), we have developed a framework that, by combining...to mutation distance and editing efficiency.   3. Optimizing "cut-to-mutation distance" for homo or...or the other on each target site.   Figure 3: Optimal cut site to mutation distances for heterozygous...PMID: 23287722. PubMed Central PMCID: PMC3712628. 3. Paquet, Dominik, et al. "Efficient introduction of...mutations When thinking further about the distance relationship described above, we realized that, in addition...efficiency already drops by half and after only about 30 bp, it is no longer feasible to incorporate mutations...Additional Resources on the Addgene Blog Learn about High Fidelity Cas9 Variants How to Design Your gRNA...

...these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8 month formal ...cover during the year. Suggest that each person bring 3 topics and write them on a board to see which ones... a regular schedule might work. If not, perhaps a 3 hour breakfast once a month. All participants must...for Scientists Series. I have been thinking a lot about Mentoring for over 10 years. Many successful scientists...Medical School researcher Connie Cepko to learn about her mentoring style. While working on this project...postdocs I have started to talk to trainees more about peer mentoring. They want to find mentors to help... questions I get is, “What are we going to talk about?” There are an infinite number of ideas on the internet...

...the limit of what will be packaged. The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase II promoter...are based on adenovirus 5 (Ad5).     Fig. 3 - Viral vector components of adenovirus Building...Proteins: Structure and Function. Viruses. 2021 13(3):388. DOI:10.3390/v13030388 Coffin J.M., Hughes S....aren’t reflected in the common components outlined above for gamma-retroviruses. While not required for lentiviral...and helper plasmids for all the viruses discussed above (gamma-retrovirus, lentivirus, adenovirus, and adeno-associated.... Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Principles of Retroviral Vector ...

...amounts to a small difference of two or three weeks. 3. How would you like to select for your flies? Sure...consumption. Drosophila are also used in cancer research (3). The sky is the limit. As mentioned earlier, even...PMID: 19563763. PubMed Central PMCID: PMC2753793. 3. V. A. Rudrapatna, et al. Dev Dyn. 241. 107–118 (2012...immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed to look...Additional Resources on the Addgene Blog Learn about Using Cre in Drosophila Read Other Genome Engineering...

...measurements; (2) the cells you’re targeting; (3) how much expression is needed to read your experimental output...gene. Off-target expression can still occur (Fig. 3) - for example, some neurons can transiently express...; Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters demonstrating...anterograde tracing system. Experimental Neurobiology, 29(3), 219–229. https://doi.org/10.5607/en20006 Rolland...Molecular Therapy - Methods & Clinical Development, 3, 16062. https://doi.org/10.1038/mtm.2016.62 Su, M....starting the selection process. Factors to think about are (1) your experimental conditions and measurements...

... transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid. These are transfected into... virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars were less stable than...with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details, check out this review... spumaviruses to human serum by galactosyl(alpha1-3)galactosylation. Journal of virology 71:6174–6178 ... also toxic at high concentrations as described above and is inactivated by the complement system in serum...

...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of 3-mercaptopyruvate...opioid use disorder,” says Hubbard. Figure 3: 20x confocal image of the periaqueductal gray (PAG...students as we have done in the past. Below, read about the five winners and their research.   Abhi Aggarwal...work has only been made possible due to the collaborative and highly interactive environment that Janelia...biosensors or biosensors that detect new redox metabolites are vital for researchers to better understand...

...-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.7] Integrin alpha-V,Integrin beta-3 Human, Mouse IgG1 Rabbit...-Integrin alpha V beta 3 [IPI-ITGAV/ITGB3.13] Integrin alpha-V,Integrin beta-3 Human, Mouse IgG1 Rabbit...roles of individual integrins. The Ha lab — in collaboration with the Springer lab, the Jaumouillé lab, the...recombinant antibodies and plasmids. To encourage collaboration and education in protein science, IPI hosts ...events and training courses on technologies and laboratory techniques related to antibody discovery. You...the Institute for Protein Innovation. She writes about all things IPI, from the scientists and scientific...

...CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC -3’; Reverse oligo: 5’- AATTGXXXXXXXXGAATAGGTCTTCTCGAAGACCCAAACNNNNNNNNNNNNNNNNNNNC -3’, where ...PMID: 26780180. PubMed Central PMCID: PMC4744125. 3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated...How Barcodes Can Be Used in Lineage Tracing Read about CRISPR/Optogentics Tools Resources on Addgene.org...

.... We have been using yeast models (3, 4) to answer questions about the evolution of the genes involved...complementation. In the procedure diagrammed in Figure 3, students first transform the S. cerevisiae met deletion...: US Government Office of Science and Technology. 3. Duina, A. A., Miller, M.E., & Keeney, J.B. Budding...Addgene Blog Learn about Synthetic Biology with BioBuilder Use Addgene Videos to Learn about Basic Lab Techniques...species, but these studies generally leave questions about gene function wide open. What if two seemingly similar...question, the plasmids are also used to teach students about restriction mapping and western blotting. Students...communicating science to the public. You can find out more about Clare at the Biology Dept. site.  References 1. ...

...acceptor fluorophore--think of it as a virtual photon 3. Relaxation of the acceptor fluorophore back to its...PMID: 27649177. PubMed Central PMCID: PMC5038762. 3. George Abraham, Bobin., et al. "Fluorescent protein...Nature 388.6645 (1997): 882. PubMed PMID: 9278050. 3. Meng, Fanjie, et al. "A fluorescence energy transfer‐based...can be done using FRET! Read on to find out more about this amazing imaging technique and find further ...our curated list of biosensors too, as another laboratory may have already created the sensor that you ...ability of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments ... Tips for using FRET in Your Experiments Learn about Fluorophore Targeting for FRET Studies FRET Based...

...used outside of the laboratory. The detection threshold for these RNA sequences (3 nM), however, was quite... samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined...PMID: 25417166. PubMed Central PMCID: PMC4265554. 3. Pardee, Keith, et al. "Paper-based synthetic gene...allowed gene circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable... quite high and above what would be expected in patient samples (e.g.: blood, urine). Adapting toehold...

...    Step 3: Reviewing the plasmid summary on the Fragmid website.   Step 3 is simply a review...of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/j.xgen.2024.100519...Destination vector (dropdown options depending on above selections) Module set (the set of components you...vectors) Target type (drop down options depending on above selections.) CRISPR enzyme (Cas9 [sp], Cas9 [other...

...Teacher, Vol. 79 No. 3, March 2017; (pp. 208-223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S....Nature, 391 (1998), pp. 806-811. PubMed PMID: 9486653. 3. Newmark, Phillip A., et al. "Ingestion of bacterially...experiments in the beginning. In most classrooms, the laboratory experience is focused on classical embryology... Biology with BioBuilder 3D Models for learning about CRISPR Resources at Addgene.org Find plasmids ...

...implement RSMs in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high copy...and promoters with buffer sequences on their 5’ and 3’ ends (for insulation). A list of promoters and terminators...hammerhead ribozyme parts are described in Nielsen et al (3) and Lou et al (4). Despite choosing proper register...PMID: 25417166. PubMed Central PMCID: PMC4265554. 3. Nielsen, Alec AK, et al. "Genetic circuit design ...certain cellular behaviors, but little is known about the order or timing of these factors. Recombinase-based... three methods of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based...Bac Vectors Can Be Used for Genome Editing Learn about Michael Koeris' Journey from Grad Student to SynBio...

...covered (to avoid drying out) while 3 ml of melted agarose are mixed with 3 ml of 2X DMEM in a 50 ml sterile...easily achieved by splitting transfected cells into 3 plates at different densities for selection). A 2%...specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings...the hood. A 48-well plate is also prepared with about 200 ul of culture medium containing serum in each...incubator, additional growth medium is added to about halfway up each well. Tips for using the agarose...with the firmer 2% agarose gel solution discussed above for picking. Many thanks to our guest blogger Iris...

...Examples of these approaches are given in Tables 3-7. 3. Making use of AAV’s selectivity for neurons: targeting...Learn more about retrograde AAVs Resources on Addgene.org Read our AAV guide Learn more about our viral...

...transgene contains a splice site acceptor followed by the 3’-end of the transgene. When a cell is transduced by...80% of muscle cells expressing mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines the...region of homology, a splice acceptor sequence, the 3’ half of the transgene. The full length gene is reconstituted...upstream of the 5’ splice donor and downstream of the 3’ splice acceptor of the transgene to help prevent ...first time AAV user Learn about how AAV is used in retinal gene therapy Learn about the parts of an AAV transfer...

...efficiency by approximately 15-fold (Chen et al., 2006). 3. Transduction efficiency depends on viral titer Both...approximately 20–50% of the packaging cells after 1–3 days of selection. A lower percentage of surviving...." Cell 113.2 (2003): 141. PubMed PMID: 12705861. 3. Chen, Sifeng, et al. "Freeze-thaw increases adeno-associated...grown at 30 °C instead of 37 °C. If you’re unsure about the integrity of your virus, perform a diagnostic...Additional Resources on the Addgene Blog Learn All about Adenovirus Associated Virus New to Virus? Check ...Check out Our Primer for Beginners! Learn All about Lentiviral Vectors Additional Resources on the Addgene.org...

...with us about adding new scientists to their labs. Read All the Posts in this Blog Series Our 3 PIs answer...'ve heard of labs where new hires spend the first 3 months reading and writing a proposal before even ...research question that people (especially you!) care about. Have you critically evaluated your plan? Have... to complete and has multiple benchmarks. Think about what your personal goals are and set milestones .... Take the time to generate new ideas and think about them critically before diving in. Research projects...

... Core hours consist of the time between 10 AM and 3 PM, when most meetings are scheduled to take place...For example, if I needed to go to an appointment at 3 PM, I could make up the hours I had missed at another...intimidating to me, as I had a hard time thinking about how I could fill this time. I was relieved to find...attend to other commitments, without feeling guilty about missing work. As long as my work was finished on...

... microscopy. Nanobodies can be labeled with up to 3 dyes, yielding similar signal strength to a standard...nanobodies are useful for colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary...PMID: 26633879 PubMed Central PMCID: PMC4755751   3. Muyldermans, Serge. “Nanobodies: natural single-domain...Control Protein Activity Learn more about protein tags Read about E. coli strains for protein expression..., if not better, than antibodies for all of the above listed molecular techniques, but they can also be...protein purification methods. Read on to learn more about nanobodies and how their structure and function ...between a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-resolution...

...selection techniques to come up with new colors (Fig. 3), such as Radish and Carrot, and further means to ...imaging in situ”. Nature Methods 9, 787–790 (2012). 3. Filonov, Gregory S., et al. “In-gel imaging of RNA...RNA-Modifying Enzymes.” Cell Chem Biol. 2016 Mar 17;23(3):415-25. PubMed PMID: 26877022. PubMed Central PMCID...target metabolite creates an aptamer that will only bind to DFHBI in the presence of that metabolite (Fig...approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up aptamers (or, fluorescing...fluorophore protein. Years later, Samie Jaffrey’s laboratory took advantage of their deep knowledge of the...Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein Activity with GFP Resources...

...effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid B (as positive control...we have successfully transfected our cells (Figure 3), we can continue with our experiment, incorporating...optimization within your experiment. Read on to learn more about the various controls that can be used for plasmid-based...: Expression Level of Gene X The results shown above are from a single experiment wherein Plasmid A (...be compared. The Empty Vector Control (mentioned above) could also serve as an important negative control...experimental cells. For example, in the experiment above, Plasmid A contains an shRNA that targets human ... times and on different days. Using our example above, we would test different preparations of Plasmid...

...possible because there are 4 bases (A, T, C, and G) and 3 positions in each codon. There are therefore 64 possible...possible codons but only 20 amino acids and 3 stop codons to encode leaving 41 codons unaccounted for....PMID: 15678157. PubMed Central PMCID: PMC1299251. 3. Emilsson, Valur, and Charles G. Kurland. "Growth ...that lead to these preferences and what can we do about them? Read on to find out!   Why do organisms have...understood, but some possible reasons include: Metabolic pressures - it takes cellular resources to produce...amino acid I’d like to encode,” but, as described above, not every region of a protein should necessarily...Effects on transcription (remember, it’s not all about translation - e.g. codon choice may interrupt transcription...

... than fusions to 3 or more HA tags. 4. High-resolutions microscopy As indicated above, smFPs can be used...moleculare were better resolved with smFP_FLAG than GFP.   3. Enhanced ability to distinguish weakly expressed ...brain". Oxford University Press, New York. pp 719. 3.Rizzo, Mark A., Michael W. Davidson, and David W. ...enable accurate labeling of neurons. As discussed above, weakly expressed proteins in particular are difficult...resembles images of the deity (see comparison image above). The church of the Flying Spaghetti Monster is ...

...fidelity of cohesive end ligation by this enzyme for all 3- and 4-base overhang sequences under standard reaction...assemblies and over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases...plate using outgrowth volumes described in Figure 3, with calculated yields from entire outgrowth built...Inserts, either precloned or in amplicon form (shown above) are flanked by BsaI sites in an orientation that...Check out NEB's work on ligase efficiency Learn about other plasmid cloning techniques Resources on Addgene.org...

... different systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion ...activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including many...Science. 1986; 231:699-704. PubMed PMID: 3080805. 3. Brent, R., and Ptashne, M. A eukaryotic transcriptional... Plasmids Read our Blog Post About Yeast Vectors Read our Blog Post About Tagging Yeast Genes ...

...program to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination ...Gateway cloning efficiency can reach >99%.   STEP 3: Express your Gene of Interest! Be sure to verify ...2003 Feb;Chapter 5:Unit 5.17. PubMed PMID:18429245. 3. Ptashne, M. (1992). A Genetic Switch: Phage (Lambda... expression systems. Keep reading to learn more about the Gateway cloning method and its advantages. An... on the Addgene Blog Try Out FastCloning Learn About Methylation and Restriction Enzymes Use Colony PCR...

...genome which was composed of the wild-type 5’ and 3’ LTRs, the ψ sequence, a part of the env gene containing...exception of the env gene (packaging plasmid), and 3) the vesicular stomatitis virus G protein (VSV-G) ... been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the best production...Science 272.5259 (1996): 263. PubMed PMID: 8602510. 3. Yasutsugu Suzuki and Youichi Suzuki (2011). Gene ...first one and can be used routinely in a research laboratory. The risk of generating RCL is low but extra ...even your own! You should therefore be cautious about inserting potentially harmful genes into a transfer...

...sequence for recognition by QCascade; although 5’-CC-3’ is one of the best PAMs for VchINT, many other PAMs..., by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple distinct targets... or isolated (Rubin et al., 2020).  Figure 3: Multiplexed insertions with Type I-F INTEGRATE using..., 2020), to multi-copy genomic integration of metabolic cassettes (Zhang et al. 2020); however, the technology...