...have the required expertise to help move your project forward? Most projects typically require a diverse...brainstorming sessions to maximize productivity and direction of the project? What is your timeline? A typical...design, execute, analyze, repeat pathway that is required of scientists.” Professor George Church encourages...approach. At Addgene, we've heard of labs where new hires spend the first 3 months reading and writing a proposal...

...can change entire culture) Specific requirements for hiring more common Likelier to hire on general...implicit aspects of today’s postdoctoral training can directly interfere with a seamless jump into industry.”... opportunities to do science related jobs that require no bench work Better resources available ($)—can...

...antibody that is directly conjugated to your reporter circumvents the issue. However, directly conjugated primary...Strategies for avoiding background secondary binding Direct immunohistochemistry (Figure 2A)  How do you avoid...species on species IHC. (A) One solution is to use direct IHC, which uses just a primary antibody that has...has your reporter (in this case a fluorophore) directly attached to it. (B) Another solution is to combine...

...showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts...mammalian cells, likely due to high temperature requirements. To solve this problem, Gao et al. searched ...Like Cas9, NgAgo is also able to mediate homology-directed repair when a template is supplied. Find Plasmids...Cas9 and the other CRISPR enzymes, NgAgo does not require a PAM sequence, so users should have much more ...single mismatch at any position of the DNA guide impaired NgAgo activity, with three mismatches abolishing...

... that we are doing just that. Do you like the direction the blog has taken over the past year? Do you ... 10 blog posts of 2015 1. CRISPR 101: Homology Directed Repair Addgenie Chari Cortez explains some of ...of the basic science behind homology directed repair (HDR) and how it’s used in CRISPR genome editing. ...vector contaminants in your cloning experiments and tired of having to PCR many colonies to find a successful...making a transition into a new job or maybe to an entirely new career path, a career coach might be able ...

...multiple genes. Modifying the CRISPRi system for multidirectional control The CRISPR interference (CRISPRi) ...Modifying the CRISPRi system for more flexible multidirectional control, a new study repurposes the sgRNA ...regulators allows a greater degree of control of desired expression levels dependent upon the type of transcriptional... E. coli. The utility of simultaneous and multidirectional multiplex control via scRNA design is readily...RNAs can be designed to allow simultaneous, multidirectional and tunable control of gene expression. ...

...Drobizhev et al. 2011). But this requires other lasers than the standard Ti:Sapphire laser. It is also important...processes during embryonic development. These are required for a growing organism to make the complicated...for many years, researchers lacked good tools to directly follow immune cells in lymphoid organs. The development...Good two-photon probes when using a standard Ti:Sapphire Laser include: Blue-green fluorescent proteins...PMCID: PMC2700268. 8. Hickman, Heather D., et al. "Direct priming of antiviral CD8+ T cells in the peripheral...

...of the fittest. Cancer cells find a way to grow haywire, access and create more vasculature to feed themselves... tumor cells that escape the elimination stage acquire genetic alterations and alter cell-surface antigen...cells would normally kill cancer cells but are directed not to through these various signals. Over-production...the microenvironment help create tolerance beyond direct proximity to the tumor; chemokine-aided activation...a variety of up and coming therapies. Future directions and open questions in cancer treatment We are... is to be successful, cancer immunotherapy must aspire to be a field of healthcare as dynamic as cancer...

...species. Administration of these dyes results in direct binding between the dye and the endogenous target...target, and thus no genetically encoded tag is required. These dyes can also be attached to an antibody ...substrate ligand motif attached to a fluorophore. When paired with a protein of interest, these tools provide...biotechnology (Grimm & Lavis, 2022). These variations directly impact the way you can utilize these dyes. The...another factor to consider. If your application requires longer periods of exposure, you’ll want a more...intermolecular FRET for ligand-receptor interactions, require specific considerations regarding the choice of...

... event. What are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital...that are released from cells during production (Maguire et al., 2012). They saw that these AAV particles...development of the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to...delivery of therapeutic proteins to the brain which requires crossing the blood-brain-barrier. In summary, ...: 28082074. PubMed Central PMCID: PMC5368844.  Maguire, Casey A., et al. "Microvesicle-associated AAV ...

...that when you click on the link it will take you directly to the original article online. It is also designed...several journals or organizations now recommend or require authors to use specific PID formats. For example...Addgene registered prefix and the Addgene ID # are directly at the end of the n2t.net web address. For the...scicrunch.org/resolver/RRID:Addgene_87155) will take you directly to the lenti-Cas9-VQR plasmid page. Despite their...your choice. Or if in doubt, copy and paste the entire line. Cite plasmid using a resolvable compact identifier...

... institute, meaning the host university does not hire or pay them (typically). A visiting scientist is...from equipment sharing or a collaboration – it requires re-location of the scientist to the new institute... reason. Visiting would allow you to train and acquire the data you need on the new instrumentation. Bonus...yourself. Identify reagents you will need, any required training, and a priority list of experiments you...have a bench mentor or anyone else available to directly help you during your visit. Reach out to your ...

...thus inactivating genes), while breaks repaired by homology directed repair (HDR) pathways can modify the...causes two types of editing outcomes: Cas9 breaks repaired by the non-homologous end-joining (NHEJ) pathway...Richardson, Christopher D., et al. "Enhancing homology-directed genome editing by catalytically active and inactive...26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human genome engineering by controlled timing ...

...scientists seek to directly probe and build new systems. Such goals require methods to directly activate and...TCS-induced responses are varied; one outcome is directed transcription from a given promoter. To harness...been greatly streamlined; only two plasmids are required per system. Applications of these systems The ...

... bacteria) or incorporate the genetic material directly into the genome to form a stable transfection....the plasmid, and can be assayed conveniently. A direct test for your gene of interest may be another method...viral-based ORIs to fill the void. These ORIs, however, require additional components expressed in trans within... Stably transfected cells may be considered an entirely new cell line from that of the original parental...important to determine the proper concentration required for efficient selection. This is usually achieved...

...for use in prokaryotes and eukaryotes, works via direct activation. In this system, the activator rtTA ...negative inducible pLac promoter. This promoter requires removal of the lac repressor (lacI protein) for...eukaryotic cell types (mammalian, yeast, plants, etc.) require unique promoters and there is very little crossover...induce a given promoter varies. Promoters that require only the addition of an external inducer, like ...very rapidly. In contrast, promoter systems that require transcription of a repressor/inducer will have ...

...very similar for both, though tissue samples do require an extra homogenization step. For this blog, we're...throughout the sample processing and therefore will require crosslinking, which uses formaldehyde or UV light...provides a truly random digestion, but is harsher and requires more optimization. Enzymatic digestion, while ...both tissue or cell type, amount of protein, and desired fragment length for your output assay. A longer...https://www.wannapik.com/   Even though ChIP requires a larger upfront investment than many other antibody...

...though. Viral packaging and entry The genetic cargo requires viral packaging to infiltrate and integrate into...outlined above for gamma-retroviruses. While not required for lentiviral production, they can be helpful...packaging proteins for AAV are rep and cap. Rep is required for viral replication and also aids in infrequent...target different tissue or cell types. AAV also requires some back up from adenovirus genes – E4, E2a, ...LXs/Iva2 are primarily capsid assembly proteins required for packaging of the virus into particles. E2A...

...use site-specific recombination, it also doesn’t require a ligation step. Based on the robust system of ...that position, preventing T4 from chewing back the entire 10–12 bases. As such, the use of LIC is often limited...the same tube. Alternatively, SLIC can also work directly on PCR products, with 5′ overhangs generated either...product with two 5′ overhangs, so these approaches require higher concentrations of insert to succeed. 5′ ...polymerase is much less expensive than the enzymes required for Gibson assembly. SLIC is a standardized method...

... is used by screen readers to provide visually impaired visitors with information about images that a ...primarily negative. Stupid, overwhelmed, discouraged, tired…words that indicated there were many barriers between...paper.  Of course, when you think about writing an entire scientific paper, you may feel it’s overwhelming...describe the image in a way that helps visually impaired readers receive the same information as sighted... writing. You can use them to help you write an entire manuscript, or you could use them to write alt ...

...often used for site directed mutagenesis. During this process, incorporation of a desired mutation into your... not the newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions...not correct! Conversely, enzymes such as DpnI require methylation at their recognition sites in order...overlapping methylation Not affected Not affected DpnI Requires methylation for activity Not affected Not affected...

... and synthetic biology. The mistrust and anger directed at GM crops has burdened this emerging engineering...Revolution Bio, we are addressing this challenge directly by developing a plant biotechnology product specifically...through conventional breeding; synthetic biology is required to achieve this novel and unique phenotype. It...control over their interaction with it, we hope to inspire and open a new conversation about GMOs.  Challenge...technologies like those pioneered by Ewen Mullins of Ireland’s Teagasc.  Plant biotechnology is full of potential...

...plasmid(s). Fields marked with an asterisk are required. If submitting plasmid(s) using a spreadsheet ...Unpublished or pre-publication plasmids" and set a desired distribution status. You can submit "Published ...general description of the deposit. Select your desired distribution status: Hold for publication: we... need; you can copy and paste your information directly into the file. Email the completed spreadsheet... sequences cloned into it. gRNAs are used for directing CRISPR/Cas9 to a targeted genomic location, while...assembled or theoretical — it does not have to be entirely verified by sequencing. Upload any support files...

...cannot induce gene editing, but it retains its gRNA-directed targeting capacity. By using a gRNA to target ...of these orthologs is key: since each ortholog requires a different PAM sequence, a gRNA designed for ...different fluorescently labeled dCas9s are efficiently directed to the proper target sequence. They succeeded ...color is specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence...in combination with gene editing. CRISPRainbow requires very short, 11-mer gRNAs, which do not induce ...

...retina, heart, and skeletal muscle. The dosing required to achieve these efficiencies was also between...mutation that causes Niemann-Pick disease type C, directly demonstrating the therapeutic potential for the...output the voltage changes in neural activity when paired with appropriate fluorescent optics. Existing red... sequences for genomic integration by homology directed repair. Using parts from this kit, the McCormick...The cofactor analog, phycocyanobilin (PCB), is required for PhyBΔ to be functional. Exposure to red light...

...that mismatch repair (MMR) interfered with the desired resolution of prime editing intermediates. Building... In PRIME-del, developed by Jay Shendure’s Lab, paired pegRNAs work with the PE2 enzyme from the Liu lab...that PRIME-del can be “multiplexed” using pooled paired pegRNAs.  Read the paper in Nature Biotechnology... strategy engineered by Wen Xue’s lab also uses paired pegRNAs targeting complementary DNA strands to ...sequence. As with other prime editing tools, the desired edits are templated by the 3′ extension of the ...

...’ve got you covered. Defining your control requirements To pick the best tool for the job, it’s important...important to know what the job requirements are. Here are the questions you should be asking. What kind ...recombinase. This is a time-tested system but does require knock-in of the LoxP sites, which is more difficult...control all the time If your experimental system requires tunable control - or maybe you’re just a control...time consuming. If your native protein doesn’t require this level of control, one of the previous options...

...scientists are acutely aware of these problems that directly affect their daily lives and future prospects....structure of the biomedical workforce. Quotes taken directly from participants (summarized in 2) touched upon...information on number of postdocs” “Postdocs are really hired to produce results, not scientists”  These comments.... Melanie Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic... at Scientific Meetings from Addgene Executive Director Joanne Kamens Learn how to be a successful manager...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...target gene in your experimental mice. This will require several rounds of breeding in which you’ll want...

...left school when she was hired full time as a Lab Technician. “I was so tired of being in school so I ...student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric Perkins, who has been... was devastating. At the same time, she has an entirely new path to discover and gives her time to give...interviewees. Fabienne Furt, Addgene’s Associate Director of Business Development, got her PhD in Plant ...