... Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. While two... two guides require two arrays for Cas9, two guides can be streamlined into a single array for Cas12a....applications. This set of new anti-FLAG frankebodies includes mEGFP, mRuby2, iRFP713, SNAP-tag, and HaloTag ...target multiple genes within a single cassette provides increased simplicity for combinatorial genetic...cost-effective alternative to commercial ladders which also provides a useful yet low-pressure teaching opportunity...less than half the size of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy...

... and transfer 100 µl of glycerol stock to each of 2 96-well microplates (identical copies, see image above...sometimes have to design new primers to sequence the important features. You can use primer design software such...such as Primer3 to help you design your custom primers. Remember, when designing a sequencing primer, you... to differ between plasmids, and selecting or designing a primer specifically to target this region, as...instructions on other factors to consider when designing your primers can be found here. Finally, you will... to Verify Your Plasmid Learn How Addgene Uses Barcodes to Track Samples Resources on the Addgene Website...

...about colony PCR in this blog post.   Figure 2: The steps of colony PCR.   Sanger sequencing Sanger...is not enough on its own to help you find your desired plasmid. In many cases, you will need a screening...you will need to sort through them to find the desired clone. Do your selection, then screen Why add a...what about identifying clones that contain your desired insert? This is where a  screen comes in. Let’s...blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid... of the information you need, so you'll want to design a custom primer. Because Sanger sequencing can ...

... would release them into the cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids...into agriculture as species-specific pesticides and insecticides, an advancement over previous chemical...prone to degradation – those are the words that describe RNA! Double stranded? Well, that’s just for DNA...also known as RNAi, are short, dsRNA molecules designed to degrade mRNA transcripts. RNAi activates the...sequence is found (typically RNA that the RNAi is designed against), RISC can repress translation and/or ... and repetitive sequence. All of the R-loops described above play distinct roles in promoting cellular...

...state-of-the-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how gender...approaches to gender equality over the past several decades: “Fix the Numbers of Women” focuses on increasing...using both sexes in basic research is excellent design), but they used all female stem cells—this was ...environmental factors. How to include sex in experimental design Recognizing the importance of analyzing sex, the... above, another excellent resource for research design is Kalpit Shah et al., “Do you know the sex of ...Corporations need to integrate these insights into product design. But eyes have been opened—and we cannot return...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...Requirement and Expanding CRISPR Beyond SpCas9 described the PAM specificity of Cas9s isolated from different..., and NGCG). Since then, the Osamu Nureki Lab described a rationally engineered Cas9, SpCas9-NG, which...Nishimasu et al., 2018). The David Liu Lab similarly described xCas9, which displayed the ability to target multiple... also high off-target activity, which may be undesirable in many applications. At the time of our first...post on choosing a Cas9, the Feng Zhang Lab had described eSpCas9, while the Keith Joung Lab had developed...

... high-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware...provide the construction plans or CAD (computer aided design) drawings of their devices, which makes it very...has allowed the community to regularly replicate designs worldwide. Distributing hardware via globally established...similar way to the biological protocol, and assembly designs based around the use of commercially available ...users outside the project to modify the raw and design files, allowing the device to take on a community-driven...of work from universities and research centers provides a potential conflict between traditional IP-based...

...and are considered “high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows for...element. This link provides a more extensive list of yeast auxotrophic markers and includes the associated...differ from the bacterial elements previously described. Why do scientists use yeast vectors? Yeast are... that come with working with prokaryotes. This includes but is not limited to: rapid growth, ease of replica...most commonly used selection markers in yeast and provides the element needed to overcome the auxotrophy ...

...cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. “A glance at genome editing with...ability to influence editing efficiency. In 2022, despite the increasing effectiveness of CRISPR/Cas9, scientists...

...gRNAs designed by the Kosuke Yusa Lab. This retroviral library was used to transduce T helper type 2 (Th2...

... enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation protocol...producer cells and purification protocols often describe a process that begins with cell lysis to release...

...and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In...have been a model organism for plant research for decades, and the biochemical pathways involved in central...

...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...cleaved precisely as predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified ...advantage over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors...the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely be adaptable...worries, I've summarized the three strategies described in this post in the graphic below - including ...

... level.     Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's ...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

...closely mimicking the future application. In figure 2 we provide an example of the comparison of the practical...tables with fluorescent protein properties and provides a quick way to compare fluorescent proteins, e.g...been used in yeast by Lee et al (2013) and in nematodes by El Mouridi et al (2017) and Heppert et al (...practical brightness. Therefore, practical brightness provides a better picture of what can be expected in a ...

...recombineering (2) to simultaneously incorporate multiple single-strand DNA (ssDNA) oligonucleotides (oligos)...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally...relaxed gRNA design parameters. To demonstrate the effectiveness of their library design, Moffat laboratory... pCAG-T7-TALEN(Sangamo)-Destination series, pCAG-Golden-Gate-Esp3I-Destination Takashi Yamamoto pcDNA-TAL-NC2...vector, and besides being used for restoring strain prototrophy, can also aid in designing self-establishing...

...IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination of the...molecules are recruited by the antibody to help destroy and remove a pathogen. Different isotypes also ...well as the genes that encode those proteins are designated by the lower-case Greek letters μ, δ, γ, ε, and...

...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus... articles reporting chromatin-related research describe the production of plasmids that might be useful...colleagues and guest bloggers have written posts describing new methods and tools for studying chromatin ...

...available in the adenovirus section below!   Fig. 2 - Viral vector components of AAV Adenovirus Adenoviruses...promoter should flank this component as well. On both sides of this promoter-gene combo are two long terminal...Laboratory Press; 1997. Principles of Retroviral Vector Design. Available from: https://www.ncbi.nlm.nih.gov/books...

...cellular responses in addition to antibody response 2) very high immunogenicity 3) and long-lasting immune...biology – they can deliver materials to specific destinations, haul a variety of cargo, and even handle a ...delivery of the actual virus to which immunity is desired. Instead, components of the virus are now delivered...