...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 ...

...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...

...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...

...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 ...

...’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...

...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 ...

...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...

...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...

...reviews our sequencing QC standards to ensure the entire team is using the same rubric and making the same...process also highlights other QC topics that may require additional attention during analysis. These include...and nucleotide populations. All of these topics require additional considerations to resolve. Sequencing...help QC scientists determine if enough reads were acquired from the MiSeq for assembly or with enough coverage...nanopore sequencing. This platform sequences the entire plasmid in one read and thus can resolve sequencing...

... 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 ...

... viral particles. Direct measurement of lentiviral RNA is one alternative to direct p24 measurement. Like...The two most common assays for physical titer are direct p24 measurements via ELISA and qPCR for viral RNA.... Developing the proper control line typically requires transducing with a limiting multiplicity of infection...labs that will routinely be producing virus or require precise titers. Additional considerations for titering...

.... In this event, the degron domain shuttles the entire tagged protein to the proteasome for degradation... additional sequence (7 kDa). This system also requires less genetic engineering than some of the other...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir.... An obvious drawback of the system is that it requires an additional component to be engineered or expressed... with additional components beyond the minimum required for a degron system, then it’s probably best to...

...a few aspects in which spectral flow cytometry requires special considerations. Conventional flow cytometry... your panel. Highly expressed markers should be paired with dimmer dyes so they don’t “wash out” the rest...Conversely, markers with low expression should be paired with brighter dyes, so they can more easily be ... two overlapping cellular markers should not be paired with these dyes, as this would create difficulty...samples stained with a single fluorophore. They’re required for every fluorophore you use in your panel, whether...

...light only kills microorganisms that it strikes directly. UV light exposure is also harmful for eyes and...around your work area. Make sure to only bring required materials into the hood. This will give you enough... avoid sticking pipettes into solution bottles directly, rather decant the solutions into disposable sterile...depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd – 4th passage...blogger, Sana Khan Khilji! Sana Khan Khilji recently acquired her Master’s degree in Biochemistry from Freie...

...AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded intermediate...work with. These plasmids contain ITRs which are required for proper packaging of AAV, but also form secondary...AAV than I think I need. Unlike lentivirus, AAV requires a higher number of viral particles for efficient...several hours on a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too...AAV for my cells of interest. Every batch of AAV requires its own MOI optimization to account for batch-...

...cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb of homology is typical for targeting...conditional knockout. This experimental design requires two rounds of recombination – the first removes... recombination can occur in multiple ways. The desired recombination event will remove NeoR only and leave...genomic sequence and create a break that can be repaired by homologous recombination using a repair template...on the Addgene Blog Get the Basics of Homology Directed Repair Read about Generating Knockouts with CRISPR...

... genome editing for a few months. More time is required for its optimization and development before it...features of NgAgo First, unlike Cas9, NgAgo does not require a PAM sequence for target recognition, which gives...Additionally, in vitro assembly of NgAgo/ssDNA requires incubation at 55 °C - a dangerous, non-physiological...regions of the genome or just after a stop codon (as required for C-terminus epitope tagging). NgAgo survey ...respondents "Final Verdict", is that more time is required for NgAgo optimization before we can make any ...

... a plasmid whose copy number is unknown, it is entirely possible that you are working with a low copy ...Start with a single, fresh colony Sub-culturing directly from a frozen glycerol stock or agar stab may ...exchange will prevent your cultures from reaching the desired density. The volume of your flask or culture tube...cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration (260... temperature. These recommendations also often require a longer growth time. Note: Julian Taylor-Parker...

...end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But...end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for...spent cloning. In contrast, the short homology required by MMEJ can easily be added via PCR amplification...genomic DNA. These three double stranded breaks are repaired by two rounds of MMEJ. This double MMEJ strategy...Resources on the Addgene Blog Brush up on Homology Directed Repair and Non-homologous End Joining Read About...

...stripes in the acquired image (5).  A way to get around this problem is to illuminate and acquire images of... of the solitary tract.  The arrows point the direction of the specimen, A= anterior, P= posterior, D=...  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow and unwieldy for...) sensors, it takes only a few milliseconds to acquire one image, thereby significantly reducing the time...

...transfer plasmids have a wildtype 5'LTR, which requires the presence of the Tat protein to work, while...'LTR. Including a chimeric 5'LTR removes the requirement for the HIV Tat protein, thus decreasing the ...gag, pol, rev, and tat. 3rd generation systems require an envelope plasmid, and two packaging plasmids...2nd-generation system because the wildtype 5'LTR promoter requires Tat to function. Third-generation plasmids can...plasmid(s) contain the minimal number of HIV genes required for virus production (3 or 4). Third-generation...

...in different CGBE tools allow the researcher to direct the repair of the abasic site. But what if an editor...transversion editors. Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate...this point relied on deaminase enzymes, either to directly accomplish the base edit or to create the substrate...they realized that MPG could be engineered to act directly on unmodified G bases, resulting in a G → Y (Y...Conclusion With these advances, every base can now be directly targeted by a base editor, although the efficiency...

...incredible versatility opens aptamers for use in many entirely new applications including aptamer based fluorescent...sensitive detection assay. In 1999, Grate and Wilson paired laser cleavage and an RNA aptamer that bound the...to induce fluorescence outside the context of a paired fluorophore protein. Years later, Samie Jaffrey...mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein equivalent... less immunogenic than proteins. Assays that require recognition of ions or particularly small molecules...

...tools, approaches, and infrastructure necessary for direct tests of cause and effect hypotheses between gene...unavailable.” As stated by one of the NSF program directors, this program should “enable the ability to study... in the tree of life.” Accepted proposals were required to present research plans that went beyond obtaining...is no small task. The EDGE program solicitation requires funded labs to rapidly disseminate protocols, ...microscope with polarizing filter. Development is direct in this species, and hatchlings are around 1mm ...