...’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...

...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 ...

...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...

...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 ...

... 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...

... 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...

...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-...

...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...

.... 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...

...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...

...created a yeast-based directed evolution approach with some notable improvements. Directed evolution techniques... fluorescent protein--no affinity purification required! The team used other molecular techniques to introduce...microtubuli and plasmids tagged with MapTau to label entire microtubules. Find these fluorescent mammalian ...contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag...

... by collections of neurons that are intricately wired into circuits through synaptic connections (Shepherd...cross-react with other antibodies, or are unavailable entirely. The other advantage is that these tags are small...intrinsic visual signal, they cannot be detected directly in live imaging experiments. It is therefore difficult... in the brain and can be detected through their direct fluorescent signals or using anti-FP antibodies...primary antibodies which can be used to amplify the direct smFP signal within sub-cellular structures. For...

...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 ...

...). These so called “plasmid addiction systems” require dividing cells to retain plasmids - if a cell does...Figure 2). This MazF-ZeoR cassette is flanked by two direct repeats which can be used for marker removal so..., knock in a GFP cassette, and introduced site-directed mutations, all without introducing unwanted selection...short-lived RNA antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation...Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of...