...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...

...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...a gene of interest is one of the most common applications of precision genome editing, the remainder of...single molecule resolution of digital PCR allows classification of individual target molecules based on their...

...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible...conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible photoactivation... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

...Golden Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to...the transformants onto the selection plate. Figure 2 shows representative transformation plates obtained...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... (percent correct) Calculated colony totals Per 2 μl assembly reaction Per full assembly reaction** ...calculated yields from entire outgrowth built from 2 µl of the assembly reaction, and from the entire assembly... bias profiling." bioRxiv (2018): 322297. 2. Potapov, Vladimir, et al. "A single-molecule sequencing assay...upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding sequence...

...Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications of the NLuc technology. The...GpNLuc and OgNLuc are available through Addgene (Cat.# 70185 and 70186, respectively). Antares Chu, J...construct for Antares is available through Addgene (Cat. # 74279). Enhanced Nano-Lanterns  Suzuki, K., et...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...fluorescence. The paper reports a wide variety of applications of the Enhanced Nano-Lanterns including monitoring...

...all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

... of 2 components: 1) an SB transposase, the enzyme required for catalysis of transposition; and 2) a transposon... gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is expressed (red stars) ... 2015 Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive... Specifically, viruses are able to infect and replicate in host cells, but they are susceptible to cells... though not all, of these defenses. For some applications of genome engineering - such as certain forms...to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion site.   The...containing the gene-expression cassette that can translocate within the genome. The Sleeping Beauty transposase...

...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...most obvious example of an individual part, this category also includes promoters, UTRs, terminators, etc...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...performing production and subsequent necessary modification of these chemical products. The flexible nature...

...is funded have been well documented over the past 2 years (Alberts et al, PNAS 2014; Taylor, eLife, 2016...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al. “Avoiding a lost generation...Riglar Advocating for Science Symposium and Workshop 2016 – Sept 16-17 MIT, Boston The Advocating for Science...futureofresearch.org/advocating-for-science-boston-2016/. Sign Up For the Advocating for Science Symposium...Director of Media Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner...union dues. Time will tell the extent of the implications of this ruling and whether graduate students...labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy of...

... in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber into .../nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of optogenetics. Annual review...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; ...

...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Options for CRISPR Genotyping Grad School Advice Part 2: Building Community Engineering the Plant Genome Using...fantastic posts in October. Click on the links below to catch up on ways to use CRISPR in plants, to learn techniques...Writing 5 Reasons to Use Reddit for Science Communication Changing Labor Laws Bring Increased Postdoc ...

...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material." Human gene therapy 21.10...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 ...

... needing to work with a transfectable cell line. 2) They require larger expression vectors which are ...are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method... Cpf1 expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from...Biotechnology 35.1 (2016): 31-34. Pubmed PMID: 27918548. 2. Zetsche, B., Gootenberg, J., Abudayyeh, O., Slaymaker...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID... a simple and effective method with multiple applications (in vitro, in vivo, transfection, viral transduction...Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background with Our Guide Pages...

...estimate transformation efficiency (Figure 2).    Figure 2: Example of a serial dilution to estimate...ask for an amplification protocol with your deposit. This is because repeated amplifications of pooled ... an amplification protocol developed for each individual pooled library; generalized amplification protocols...Plasmids Variable Region Size DNA per Amplification Amplification Type Link to Protocol CRISPR Knockout... this blog, we’ll discuss how to develop an amplification protocol for your pooled library deposit. When...to amplify the sample for further downstream applications. As individual libraries can vary significantly... to consider when creating a pooled library amplification protocol. Created with BioRender.com.   ...

...Topic) 0:00 - 2:00 : Introduction to the podcast from producers Tyler Ford and Eddy Page. 2:00 - 3:55 : ...MD PhD process and explains why he loved the first 2 years of medical school. 7:05 - 10:55 : Alex's focus...materials (check out our new Viral Service), and educational outreach. Many thanks to Alex Chavez for taking...

...below. Types of Voltage Indicators GEVIs can be divided up structurally into 2 main groups: voltage sensitive...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...Voltage indicators are voltmeters for cells! In this blog we will review how these indicators work, their...fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously mentioned...Enhanced genetically encoded voltage indicators advance their applications in neuroscience. Curr Opin Biomed...Cellular communication and function depend on potential energy. Cellular activities like action potentials...their general applications, and why viruses are essential to their use as biological tools. Why Use Voltage...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...run in triplicate.  The nice thing about qPCR is that since it measures relative quantification - that... not need to be run in triplicates. ddPCR is an extremely accurate application, even in areas of the genome...Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications Resources on Addgene.org...blog post, we're going to focus on PCR-based applications (qPCR and ddPCR.) qPCR Quantitative/real-time...range of RQ values as noted above.   In both applications, results can be affected by DNA quality and ...

.... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed... I hope these tips demonstrate that plasmid verification is not just necessary but also an easy process.... Please vist Addgene's resource for plasmid verification to find additional tips and detailed protocols...

...help in multimer identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...