...approximately 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically...expressing a novel reverse transcriptase (RT), MarathonRT. Reverse transcriptases are critical tools for ... to transform and express in bacteria, which, in turn, makes them more adaptable to a broad range of applications...!  MarathonRT: A highly processive reverse transcriptase Article contributed by Will Arnold  Listen ...RTs in this regard and is even able to reverse transcribe the extremely long, highly structured HCV genome...structured template!). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other... are dependent on long, error-free, reverse transcription of RNA such as long-read cDNA sequencing and...

... for studying translation in various cell types and physiological states. References 1. Ingolia, Nicholas...various stimuli on global translation, but a visual technique to study translation remained elusive. Two ...easier to track translation in two different ways: by monitoring the first round of translation or by tracking...destination co- or post-translationally. TRICK: Visualizing the first round of translation While ribosome profiling...in the fluorescent signal: untranslated RNAs appear yellow, and translated RNAs appear red. To enable ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is necessary...readout of relative reporter translation. Single molecule continuous translation tracking TRICK allows you...

... handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2...reproducibility As Jeffrey Flier points out in his talk (1:24 - 29:31), there are a variety of ways of defining...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also initiatives to create more ...found. Consider the hypothetical situation in Figure 1 where many scientists are performing similar experiments...using the actual tool that you ordered and not a random aliquot pulled from a freezer. As you’ll see in...

...Tris-tricine 2.5–40 kDa Tris-tricine 30 V, 1 hour or 100 V, 1–2 hours Good separation, quality, and stability...Limited protein size range Tris-acetate 40–500 kDa Tris-tricine 150 V, 1–3 hours Good for higher...detection quickly (~1 minute), and have a linear response over a broad dynamic range that typically spans...lot to cover, so let's dive in!      Figure 1: The technical decisions to make when designing a ...assay. 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...weight of the proteins you’re interested in. Table 1 lists the most common types of gels for SDS-PAGE running...

...the range of the assay for a wide range of titers. We usually dilute our samples 1:6 million to 1:25 million...important to note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies (GC) per ...In the image to the right, there is approximately 1 positive copy per microliter in the no template control...elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation...diagnosis." Emerging microbes & infections 7.1 (2018): 1-9. PubMed PMID: 29691363. PubMed Central PMCID: PMC5915492...determine if your titers are within the expected range. We have also found that generating the standard...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...

... 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a genomic site specified by a guide...tag. The two PCR reactions are then mixed and transfected along with a Cas9 expression vector. They can...

...materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created ...Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells. Methods Mol Biol. 2018;1850:1-16. doi...Neuroscience Research. Curr Protoc Neurosci. 2020 Dec;94(1):e106. doi: 10.1002/cpns.106. PMID: 33151027; PMCID...doi: 10.1007/978-1-4939-8730-6_1. PMID: 30242676. Frenzel A, Hust M, Schirrmann T. Expression of recombinant...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...mammalian cells can perform human-like post-translational modifications that bacterial and most yeast ...be produced through a single or dual-plasmid transfection, depending on whether the heavy and light chain...

...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...References 1. Kowarz E, et al. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell...important for social and regulatory reasons. 1.) The transposon consists of a mirrored set of inverted repeats...T cells to target CD19. Immunol Rev. 2014 Jan;257(1):181-90. doi: 10.1111/imr.12137. PubMed. Find the ...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...

...For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one expressing Cpf1 and the other...approaches have two main drawbacks: 1) Most rely on transfection of more than one vector to express the...comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1 CRISPR Nucleases   ...leave them in the comments below.        References 1. Zetsche et al. "Multiplex Gene Editing by CRISPR–...the usual transfection drawbacks: transient expression and needing to work with a transfectable cell line...Cassettes transfection gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9...Cassettes transfection gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9  ...

...critical first step of a successful transduction experiment. References 1. Kutner, Robert H., Xian-Yang Zhang... for predicting gene transfer events." Genetic vaccines and therapy 2.1 (2004): 1. PubMed PMID: 15291957...type of assay is a clonal cell line known to contain 1 integrated copy of the qPCR target gene. Developing...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009... titration methods."BMC biotechnology 6.1 (2006): 1. PubMed PMID: 16836756. PubMed Central PMCID: PMC1534021...project forward, transductions should not be rushed into. Before you start any transduction, you should always...Primers targeting the transgene are also beneficial as they ensure that the correct transfer plasmid was used...

...across scientific disciplines. Scientific Data, 8(1), 1–11. https://doi.org/10.1038/s41597-021-00981-0 Wright...excellent at infecting our target neurons (Figure 1). However, this one set of experiments didn’t provide...yet none of that data was published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8...purification and formulation. Molecular Therapy, 12(1), 171–178. https://doi.org/10.1016/j.ymthe.2005.02.021...Stimulation. Frontiers in Human Neuroscience, 14(December), 1–12. https://doi.org/10.3389/fnhum.2020.578687 Additional...expressing neurons and GFP (green) to label neurons transduced by each serotype. Working at Addgene has ...answers hundreds of AAV questions every year on a range of issues related to AAV vectors, many from people...

..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...antibiotic resistance using multiple approaches. Major restaurant chains including Chipotle and Panera Bread have... joining machinery, so a CRISPR-induced double-stranded break (DSB) in the genome is lethal. If this DSB...

...References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology: Advantages and Drawbacks. Clon Transgen 4:138...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few different...could use a vector like pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction...sequencing or restriction digest! Then, you can transform or transfect the cells that you want to use for your...

...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...jam packed with everything needed for synaptic transmission, are right around 250 nm, making it difficult...pre-expanded samples can sometimes take on a fragmented appearance post-expansion, but that doesn’t mean we should...brief history and new avenues. Philosophical Transactions Royal Soc 380:20210110. https://doi.org/10.1098...

... an envelope are called enveloped viruses (Figure 1). Like bacteria, only a tiny fraction of viruses are...are pathogenic (Balloux & van Dorp, 2017). Figure 1: A non-enveloped virus (left) and an enveloped virus...create far safer viral vectors that can be used in BSL-1 or BSL-2 labs. Viral vectors are viruses that have... https://doi.org/10.1016/B978-0-12-800947-5.00004-1 Taylor, M. W. (2014). What Is a Virus? In M. W. Taylor...reverse transcribe their gRNA into DNA, which is then transcribed into mRNA before being translated into ...genome types that can be either double stranded (ds) or single stranded (ss) (ie, dsDNA, ssDNA, dsRNA, positive... of virus. RNA genomes can be transcribed into mRNA, directly translated into proteins, or, in the case...

...Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed PMID: 26702451. PubMed Central PMCID... 2005 [1]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac...integration [8].   Figure 1: Mechanism of the piggyBac™  transposase/transposon gene modification system...limit [1]. The piggyBac™ technology can be used for numerous applications, including transgenesis, gene-trap...The predicted frequency of ‘TTAA’ in the genome is 1 in every 256 base-pairs of DNA sequence, making it...technologies and strategies for their projects. References 1. L. E. Woodard and M. H. Wilson, “piggyBac-ing models...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...

...including yourself as the researcher.   Figure 1: Separation of retroviral packaging components onto...these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar to AAV in that...lab coats are either recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus...required whenever there’s a splash or spray risk in BSL-1. In BSL-2 labs, eye protection is required any time...isopropyl alcohol, 0.5% peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may...looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate... plasmid). Viral production then proceeds by transfection of all three plasmids into a producer cell line...

...coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003):...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site will...genOway press release for more information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of...infected and would have the potential to express the transduced reporter gene. Generating a FLEx switch to control...

...buffer at temperatures ranging from 25-80 °C. Activity is expressed as Kcleave (min-1). SpCas9 has maximum...Cells? Yes Yes Longevity in Serum?* No Yes Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 ...GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9...outperformed SpCas9 at high temperatures. See Figure 1 for a summary of the experimental results. GeoCas9...). G. stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature...or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70 °C Genome...Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and...

...HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central PMCID: PMC6610143...fused to a fluorescent protein. “Frankenbody,” like the infamous frankenstein, was constructed by grafting...called this probe the HA frankenbody (Zhao et al., 2019).  To develop HA frankenbody, the Stasevich lab tested...can you do with the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound...also proved that HA frankenbody can be effectively used to track single mRNA translation in U2OS cells. HA...acids, thus it is translated quickly by the ribosome. This feature provides HA frankenbody the ability to...properly once inside cells.  Developing the HA frankenbody To combat the issues with these antibody based...