...total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided high...post-transfection. Remove media, replace with fresh media. Day 3-4 (am): Harvest virus Equipment Class II, Type A2 ...obtaining high viral titer. 293T cells should be split 3 times a week: Monday: Plate 1×10 6 cells in a T75 ...enough PEI such that the ratio of μg DNA:μg PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid...DNA μL of 1 mg/mL PEI 1:1 18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4...lentivirus can be used for a variety of downstream applications such as stable-cell line generation. Workflow...without calcium or magnesium, Corning 21-040-CV (cations can affect the attachment of adherent cells) 0.45...

...pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...unstable and lack precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade...

..." Gene therapy 6.7 (1999). PubMed PMID: 10455443. 3. Hindson, Benjamin J., et al. "High-throughput droplet...the other to guess a category by listing off things that fall into that category? Okay, let’s play. I’...’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot ...concentration of viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle...value, physical titers don’t necessarily give any indication of the infectious titer. The infectious titer...affected by many experimental factors. To list some complicating factors for quantifying AAV: qPCR efficiency...optimized our assay in a few ways:  Absolute quantification by qPCR requires generating a standard curve...

...of amino acids. Each amino acid is encoded for by 3 nucleotides termed a codon. As there are only 20 natural.... Uracil replaces thymine in RNA molecules. Every 3 nucleotides (codons) in a DNA sequence encodes for...Reference Page Plasmids 101: How to Name Your Plasmid in 3 Easy Steps Molecular Cloning Techniques Reference ... Origin of Replication ( ori ) DNA sequence which directs initiation of plasmid replication (by bacteria... Educational Resources Molecular Biology Reference Molecular...plasmid selection DNA sequencing for plasmid verification Resources Genetic Code Webpage and Blog References...Techniques in chemistry enable isolation and purification of cellular components, such as DNA, but practically...

... level should now be automatically adjusted every 3 years. In May 2016, Secretary of Labor Thomas Perez...Labor’s general guide to the effect on higher education, you can see that those in teaching roles (such...frankly quite surprising. Consortia of higher education institutions submitted letters to the Department...centrally administered, which leads to many complications, including the inability of some institutions...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...of the postdoctoral workforce. Based on our communications with and research on these institutions, we...to the new level:  We are still waiting for an indication of what (most of) Harvard, Johns Hopkins, (most...

...types can be found on Addgene’s website. Fig. 3: Cas9 repressors include dCas9 alone and a dCas9-KRAB... than ever to make precise, targeted genome modifications. Cas9 has been modified to enable researchers...instead.  Choosing a Cas9 for site-specific modifications Site-specific deletions and knockouts Some experiments...mutation at a specific site e.g., deleting the catalytic residue of a protein or knocking out a very small...flexibility as to where your sgRNA needs to be located and there may not be a NGG PAM site anywhere to... Beyond SpCas9.” Single base pair edits Is the location and type of edit you need to introduce highly ...Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic a patient mutation...

... PMID: 14704360. PubMed Central PMCID: PMC373311. 3. Kim, Jung-Sik, et al. "Epitope tagging of endogenous...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,... recommends designing homology arms with the modification of interest, ordering them from a gene synthesis...with a homologous repair template. It is this application - the creation of sequence-specific knockins ...clones, making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture...tissue culture based approaches that require modification of only a single allele of a gene, such as the...efficiently deliver large repair templates for applications that require large gene fusions. The current...

... remember that antibodies recognize very specific 3-dimensional epitopes. Different sample processing ...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...will share their findings either through their publications or as a review on a company website. It is important...Resources on the Addgene blog Learn about research applications of antibodies in Introduction to Antibodies ...

...Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3. Drexler, H.G., Uphoff, C.C. “Mycoplasma contamination...positively charged polycation, polybrene. Interestingly, one study found that alternative polycations such as DEAE-dextran... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...lentivirus may want to consider testing a variety of cation and serum sources to ensure the best possible transduction...establishes the best conditions for specific applications, stick with them! Changes in such variables ...efficiency of lentiviral vectors: effect of sera and polycations.” Molecular Biotechnology 53.3 (2013). 308-14...

...biotechnology 16(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking... gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ...37 °C). Smart molecular evolution led to the identification of FLPe which has a temperature optimum of ...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...

...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase base ...2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell...to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing. Molecular...Harris RS (2018) A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9...

...Sciences, 24.9 (1999b): 359–363. PubMed PMID: 10470036. 3. Said, H. M. “Biotin: The forgotten vitamin.” American...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... carboxylase and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...uses of biotinylation in the lab are protein purification and immunoprecipitation experiments. Biotinylated...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...

...thomas-kilmann-conflict-resolution-model/, accessed Jan. 3, 2022.     Additional Resources on the Addgene blog...conflict is not always what was communicated but how it was communicated. The next step is to come up with...of lapsed communication. Making some assumptions about how something should be communicated is necessary...skill in and of itself. Conflict can also be categorized as either “personal” or “substantive”. A personality-based...traced back to one of a few trouble spots:  Communication (or lack thereof): Information sharing gets ...organization gets, and creating new avenues of communication can create as many problems as it can solve....meeting? Different teams frequently prefer to communicate in different ways, so the source of the conflict...

...recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab...activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted nucleic acid. To...amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-RPA, respectively...RNA for subsequent detection by Cas13. This amplification step in combination with the ssRNA reporter ... introduced in 2017 so the lab made further modifications to improve their detection system. They named... SHERLOCKv2 uses far less primer in the pre-amplification step allowing for greater quantitation without...hour. Find the SHERLOCK plasmids at Addgene! Applications of the SHERLOCK detection system The Zhang lab...

...predict which one will work as illustrated in figure 3 (Mastop et al., 2018). Unbiased, random insertions...study its function is one of the most popular applications of fluorescent proteins. These fusion proteins...steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites or motifs that...that are necessary for post-translational modifications. Despite these potential issues, GFP has been used...linker sequence short In the early days of GFP applications, many were concerned with steric hindrance by...N-terminus of the donor fluorescent protein can be truncated to increase the FRET efficiency. Creating fusions...fluorescent protein and protein of interest (POI). To indicate the sequence, people often use terminology like...

...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, ...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...DNA fragments contain these sites at the correct location and NOT in the middle of your insert. Gateway ...

...without the risks associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can...decades. Widely used across various research applications, they have become indispensable tools for manipulating... easily, without the need for time-consuming purification processes, and are powerful, due to their ability...-lasting changes in cells. However, for some applications, integration might be considered a double-edged...dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...

...base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. ...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

...Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294 Wang,...studying genetic modification for their ease of manipulation and their ability to be replicated. Because E...., 2004). Additionally, breakthroughs in gene modification tools and methods such as CRISPR have vastly...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef... bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899...

...protein." (1998): 509-544. PubMed PMID: 9759496.  3. Buckley, Anthony M., et al. "LOV-based reporters ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...an additional protein module that can report on location. This approach enables the study of proteins in...fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...C-terminal consensus sequence (-CaaX). Both modifications are disrupted when GFP is fused to these sequences...