... maintain itself within host cells.  The plasmid has multiple ori that are functionally differentiated...various hosts (Pagatto and Dillon 2001).  The plasmid has its own replication initiation factors and proteins...the host. Host range depends on whether the host has the components to recognize the ori and allow for...restriction enzymes (Meyer et al. 1977). So if a plasmid has few restriction sites, it may be able to sneak past...commonly used bacteria in lab manipulations. Table 1 has a detailed list of the bacterial species these plasmids...

...Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use in mammalian...biology surrounding a particular gene (although it has been quite successfully adapted for use in screening... method for validating your selected gRNA’s, and has the bonus benefit of supplying you with primers for...sturdier than the delicate RNA and therefore there has less chance of contamination with RNase leading to...products will give specific details on what exactly has been inserted or deleted. Each lab will use a slightly...

...  Disadvantages Every rose has its thorns and every affinity reagent has its downsides. These reduced-in-size...created in lab in situations where a suitable antibody has been identified but a smaller or less immunogenic...using combinatorial libraries, and high specificity has been reported for a number of targets. Due to their...

...developing and optimizing biosensors in the lab. He has recently developed a third-generation intensiometric...release from hundreds of synapses at once. “This work has only been made possible due to the collaborative ... at the German Cancer Research Center (DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based...Qiu’s work on the PV neurons is in progress, she has validated the dual virus method to study CR neurons...

... and serotype, respectively. For example, AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors... components      Cargo capacity Empty rAAV has a cargo capacity of ~ 4.7 kb, meaning that your gene...usually quite stable and in some mammalian tissues has shown expression for several years (Wonjo et al.,...scAAV vector with half the size of the ssAAV genome has a mutation in the terminal resolution site (TRS) ...

... is an amazing service to biology scientists and has saved countless hours of lab time (in both donating...have at least one plasmid deposited at Addgene that has been distributed more than 100 times.   “I really...ready-to-use viral vector preps. Today, that number has grown to over 114,600 viral preps. And in 2025, we...resources like this are why my brand of neuroscience has exploded. Because we have a resource like Addgene...

...nuclease activity, catalytically dead Cas9 (dCas9) has many applications, the most well-known being transcriptional...transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently label a sequence...imaging time for this technique. The NIH Common Fund has made mapping the 4D nucleome a specific priority,...

...Addgene has distributed over 800,000 plasmids since 2004. In that time, our lab has gotten a lot experience...

... population level, but each individual cell only has a maximum of one gene knocked out. In this genetically...containing up to around 200,000 different gRNAs. It has been suggested that researchers should use ~400X ...than gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the... If you are lucky and find that a druggable gene has a central role in your system, you could majorly ...guest blogger Fredrik Wermeling! Fredrik Wermeling has a research group at the Centrum for Molecular Medicine...Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest in using molecular tools to...

...promoter of the 5’LTR has been deleted to reduce its activity. A CMV or an EF1α promoter has been inserted in...The risk of generating RCL is low but extra care has to be taken especially if you are working with proto-oncogenes...previously used to drive expression from the LTRs) has been removed, Rev (which facilitates nuclear export...expression. In contrast, in the Tet-on system, tTA has been modified (and renamed rtTA) to bind TRE only...viral binding. This method, called spinoculation, has been found very effective for T cells transduction...

...It is fair to say that CRISPR has revolutionized molecular biology and has enabled functional studies in...enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these...knockout methods of the same gene. However, recent work has called this prevalent assumption into question. Specifically...multiple target sequences are concatenated. hpRNA has been extensively used in plant research and many ...constructs are available to the community. Each construct has unique features, such as inducible expression of ...

... techniques is a speculation of yesteryear which has been realized recently. Science Fiction (SciFi) writers...even before the discovery of genes themselves.  How has scientific progress in genetic editing impacted the...technology on various non-scientific platforms, CRISPR has also transcended into the world of SciFi. This is...and most important genres of literary fiction. It has always served as a vision of the future and to make...and agriculture, its portrayal in popular fiction has been largely negative. But these negative depictions....1126/science.1258096 Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2018) Publisher...

...industry just wasn't for me, and as has been highlighted recently it has become more difficult to attain ...interesting and luckily genetics is a sector which has been rapidly growing. These factors left me optimistic...because it is a small and dynamic company and so it has the flexibility of a ‘start-up’. I was surprised ...schedule.  I really love my highly varied role, it has allowed me to travel and to share my passion for ...everyone. My career path may seem quite unusual but it has allowed me to try out working in many different sectors...

...Automating bacterial transformation One protocol that has been automated is high throughput transformation....benefits of the Biomek FxP is it’s customizability. It has a large deck that can house many different types ... how long to keep it there. In addition, the FxP has two robotic, mobile pods - one pod can pick up 96...cap sealer. This is a simple table top device that has a roller to go over caps and push them into micronic...

... development and function. To date, gene therapy has been used to treat 10 infants with this disease. ... nucleic acid cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies...AAV is not associated with any pathogenicity and has a low immunogenic profile. AAV was first discovered...Harshana S De Silva Feelixge is a researcher whose work has focused on gene therapy for viral infections. She...

..., full spectrum flow cytometry (“spectral flow”) has emerged as a particularly useful tool for those wishing...for PE. In order to correct for this, compensation has to be applied to “subtract” this spillover and correctly...they can more easily be detected. Cytek’s website has a convenient tool, called the similarity index, which...beads or cells. Overall, spectral flow cytometry has the ability to generate incredible amounts of data...

...   Fig. 1: SpCas9 has sparse PAM coverage in the human genome. SpRY Cas9 has PAM sites covering the... a PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there ...alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to be less effective at NGH PAMs, though...

..., that informs us that this antibody has specific binding and has a strong signal. We call the unique ...strong signal has a shorter exposure time than the negative control, and a weak signal has a longer exposure...

... transcriptional read-through. Chris Voigt’s lab has characterized a set of prokaryotic terminators and...for eukaryotic termination are understood and it has been noted that each eukaryotic RNA polymerase terminates...combination with certain viral packaging systems has been associated with reduced viral titer, but improved...Expression Levels of Lentivirus-Delivered Transgenes but Has the Potential to Reduce Viral Titer in a Promoter-Dependent...

...to see if anyone has published a protocol that would work for your system. Addgene has depositor submitted...Sep 30, 2020 by Marcy Patrick. CRISPR technology has been widely adopted for genome editing purposes because...Mammalian CRISPR expression systems Each model system has its own best practices for efficient delivery of ...