...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis... and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair. This method...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing...template plasmid, these PCR products are directly transformed into bacteria expressing recombineering proteins...fine, especially using plasmids in the 10-12 kb range. REPLACR-mutagenesis also requires making your own...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...dynamic range, and resistance to photobleaching has made luciferase a common choice in assays ranging from... luciferase. Transcriptional Element Screening Optimized STARR-seq ( S elf- T ranscribing A ctive R egulatory...including transcriptional reporters for NF-kb, TGF-b, c-Myc, p53 and MAPK/JNK transcriptional reporters...collection of luciferase plasmids for assays of transcriptional regulation and bioluminescent reporters. Plasmid...bioluminescence found in a diverse number of organisms, ranging from bacteria to aquatic animals to insects. Luciferase... protein half-lives, making them useful as transcriptional reporters. However, they are not secreted, ...

...type, and/or desired event.   Figure 1: Transduction to expression process. A viral particle enters...triple T’s of biology are transformations, transfections, and transductions! In this blog we will discuss...intricacies of transductions, we've got you covered there too. What is a transduction?  Transduction is the ... experiment. Using transduction vs. another method Transfection and transduction seem to achieve similar...picky when it comes to transfection/transduction methods. Some ‘difficult to transfect’ cells lines are amenable... process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar ...to find out more! Molecular basis of transduction Transductions utilize the ‘normal’ mechanism of viral...

...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline...testing transcription factor binding to reporter plasmid pH3U3-zif268 (omega) . Transcription factors ... answer many interesting questions. Does my transcription factor bind to a particular sequence? Do these...wild-type Cas9 from S. pyogenes for inducing double stranded breaks. For a more comprehensive list of our bacterial...see here . pGRG Series Various Plasmids Tn7 Transposition Nancy Craig A gene of interest cloned into one... one of these plasmids can be transposed into the attTn7 attachment site found in many enterobacterial...enterobacterial genomes. The bacteria of interest are transformed with the appropriate construct, grown at the permissive...

...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...control and is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...effects of the transfection itself (i.e., the vector used in transfection, transfection reagent, or transfection...

...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will...to additional protocols and plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "...

...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...