... let the panelists introduce themselves. I do a 1-2 minute introduction. If you chose good panelists, ...take 5 minutes to introduce themselves and, voila, 1/2 of your time is gone with no interesting discussion...introductions. The moderator asks questions, calling on 1-2 panelist for each question in a way that elicits those...shift, they are checking out. If you can, plant 1-2 good questions with trusted audience members. A good... about making sure the audience is interested, educated and talking as a result of the content. Here are...contribute. The moderator should identify the funny, provocative or interesting stories that will make for a fun...sure all your panelists have the correct date, location and parking information and remind them about ...

...manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification     Now that you’ve cut...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...containing the ligation mixture with backbone alone; 2. Transformation containing ligation mixture with insert...colonies than the vector + insert plate (unsuccessful 2), or roughly equivalent numbers of colonies on each... or equivalent to backbone + insert, unsuccessful 2 and 3 above), you can try ligating the recipient plasmid...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...

...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...

...and a source of sdAbs (Figure 2) (Xu, et al., 2021).      Figure 2: Genetically modified mice offer... from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature 595:278–282. https://doi.org/10.1038...composed of camels, llamas, and alpacas (and their undomesticated counterparts) - not exactly species that jump...to sdAbs becoming valuable tools for diverse applications. For example, RANbodies can be used as an alternative... excitement around using sdAbs in diagnostic applications. For example, fluorescently and radioactively...heavy-chain only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable...inspired this versatile family of tools, the applications they have already enabled, and the discoveries...

...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

.... The most common options are 4 °C overnight or 1–2 hours at room temperature (RT). Most people choose...overnight at 4 °C and their secondary antibody for 1–2 hrs at room temperature, which allows them to reduce...Tris-glycine 6–400 kDa Tris-glycine 100 V, 1–2 hours Easy and cheap to handcast Short shelf-life...–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability ...separation of proteins of different sizes.  Table 2: Recommended gel percentages for various protein sizes...are consistent and stable at room temperature for 1–2 years and are available in formulas specific to your...response over a broad dynamic range that typically spans 2–5 orders of magnitude, which includes a wide dynamic...

...or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org/10.1038...due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you ...efficiently during infection. Journal of Virology, 70(2), 721–728. https://doi.org/10.1128/JVI.70.2.721-728.1996...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...

... although we used different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer...illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates that the usefulness of...were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries, categorized by function...insertion via a chemical modifier. We used these categories to see what kinds of CRISPR tools people were...Cumulative number of deposited CRISPR items, categorized by CRISPR function, by year. (B) Number of requests...

...substrate (see figure 2).          The artificially evolved adenosine deaminase catalyzes the conversion of...editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular components...available for plant systems. Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful...based CBEs are known to deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer....PMID: 27096365. PubMed Central PMCID: PMC4873371.  2. Nishida, Keiji, et al. "Targeted nucleotide editing... optimize base editing efficiency based on the location of the ‘A’ relative to the protospacer adjacent...Choose a base editing platform depending on the location of your target base in the protospacer sequence...

...two categories: 1) studies focused on better understanding the mechanisms of reprogramming, and 2) studies...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...for generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed PMID: 22767332.  3. Kim, Dohoon...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...authors fragmented Cas9 into N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...Starting with libraries and a biotinylated (for purification purposes) protein target of interest, the protocol...-30) of sybody candidates are sequenced for identification. All unique sybodies can be subcloned into ...2020) Generation of synthetic nanobodies against delicate proteins. Nature Protocols 15:1707–1741. https...

...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human... reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes... dissociation rates and discovered that the catalytically active form of the nuclease dissociates very...we measured identical dissociation rates for catalytically inactive dCas9 molecules. We followed these ...Our in vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure...

...PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...which provides the AAV replication (rep) and capsid (cap) genes. AAV replication uses the host’s polymerase...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...

...primer design (Figure 2), IVA cloning can be used to perform any plasmid modification, from insertions, deletions...genes. Primer design for each type of modification is shown in Figure 2, and is discussed in more detail below...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...’ Check your amplification by gel electrophoresis. In our experience, good amplification after PCR means...

...Hub, 76% of respondents indicated they wanted to see more negative data (Figure 2.) At the same time, when...descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating vector...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative...caudal NST. (A-C) Low-magnification images showing viral spread. (D-F)  High-magnification images immuno-stained... right AAV for your system or to successfully replicate published works. Of particular importance to these...performance. Yet, most of the technical data needed to replicate and build on others’ results is available not ...

...vectors: progress and applications. Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment...many CRISPR applications, and they’ve also had success in clinical gene therapy applications. Read on to...to learn more about the current (and future) applications of lentiviral vectors! Lentiviral vector history...original system, the possibility of creating a replication-competent virus via recombination between the...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,... the corresponding catalog item (see maps section for images).     Figure 2: AAV Pro cells were...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...

... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...insert comes from using Taq polymerase for the amplification step since Taq polymerase leaves a single deoxyadenosine...rejoining supercoiled DNA ends to facilitate replication. The TOPO technique specifically uses Vaccinia...Addgene.org Find More Plasmid Cloning Protocols Catch Up On Your Basic Molecular Biology Techniques Watch...

... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey... Aptamer biosensors are used in a variety of applications, including detection of environmental pollutants... opens aptamers for use in many entirely new applications including aptamer based fluorescent biosensors... render this approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up...