... both substantial merit and national importance; (2) The foreign national must be well positioned to advance...outstanding achievements in your specific academic field, (2) that you have three 3 years of research experience..., Casseus Law suggests a mix of experts including 2 dependents –people who have worked with you i.e. your... issues.  The permanent residence “green card” process is a necessary and often overlooked part of career...start planning for your visa and or green card process well before addressing your career alternatives... benefit from the applicant thinking about the process early, keeping good records, and planning for the...organizations do not have the April 1 H-1B queue process or quota like industry, these organizations can...

...PMID: 25386923. PubMed Central PMCID: PMC4227841. 2. Young, Carissa L., Zachary T. Britton, and Anne S...

...expected plasmid features.  Figure 2. Addgene’s basic QC process. Created with BioRender.com. Once...quality control (QC) processes to screen for and reduce these errors. These processes allow us to have a...developed a process for identifying, categorizing, and resolving a variety of QC issues. This process ensures...discrepancies. While Addgene’s QC process adds time to the deposit process, we believe this to be non-negotiable...research, these errors can affect experimental processes, results, and subsequent conclusions. Being vigilant... edit, and easy to share. However, the cloning process — including the propagation of plasmids in bacteria... [email protected] to get started! Addgene’s QC process Addgene prioritizes the sequence quality of the...

...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some... gene expression: Implications for gene therapy. Proc Natl Acad Sci U S A, 95(19), 11377. https://doi....Undergoing Spontaneous Degeneration in Tissue Culture. Proc Soc Exp Biol Med. 84(3), 570–573. https://doi.org... was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response...response to Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature...encode proteins essential to initiating the whole process – such as the viral DNA polymerase – it makes sense...

... “Ligand-Regulated Site-Specific Recombination.” Proc. Natl. Acad. Sci. USA 92(June): 5940–44. PubMed ...Using a Ligand-Dependent Chimeric Cre Recombinase.” Proc Natl Acad Sci U S A 92(15): 6991–95. PubMed PMID...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...Sternson. 2008. “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range ...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl...

...genomes. Nern A, Pfeiffer BD, Svoboda K, Rubin GM. Proc Natl Acad Sci USA. 2011 Aug 23;108(34):14198-203...Allak B, van Drunen E, Kanaar R, Berns A, Jonkers J. Proc Natl Acad Sci USA. 2001 Jul 31; 98(16): 9209–9214...CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin’s work shows that the ...

...Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a white...

...topoisomerase I in Escherichia coli is sequence specific."  Proc Natl Acad Sci U S A.  1991 Nov 15;88(22):10104-8... 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...restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the...insert, the magic of TOPO cloning happens. Basic procedure Let’s break down the steps needed for TOPO cloning... everything you need for the next step before proceeding. Pre-warming your antibiotic-containing plate...

...Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T. Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7....resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...

...) by temperature-sensitive Sendai virus vectors. Proc Natl Acad Sci U S A, 2011. 108(34): p. 14234-9. ... temporal modulation of canonical Wnt signaling. Proc Natl Acad Sci U S A, 2012. 109(27): p. E1848-57....revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central...

...mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51.... hybrid derived from the trp and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed ...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional...over the TetO elements, allowing transcription to proceed (pJKR-H-tetR from the Church lab uses this system...Response Element (TRE)), allowing transcription to proceed. When tetracycline or one of its derivatives is...

...piggyBac transposase tools for genome engineering.,” Proc. Natl. Acad. Sci. U. S. A., vol. 110, no. 25, pp...provides a simple tool for conditional cell ablation.,” Proc. Natl. Acad. Sci. U. S. A., vol. 105, no. 38, pp...without leaving behind any accessory DNA sequences [2], [3]. Simply put, the piggyBac™ transposon containing...with a Footprint-Free™ genome edit.   Figure 2: Editing DNA using the piggyBac™ transposon selection...PMID: 26211958. PubMed Central PMCID: PMC4663986. 2.  A. M. Singh, V. V Adjan Steffey, T. Yeshi, and D...to transfect, select, and isolate clones. This procedure is cumbersome and inefficient, as it relies on...incorporated, and used during the differentiation process. Moreover, a splinkerette-PCR assay may be used...

...sensitize and kill antibiotic-resistant bacteria.” Proc Natl Acad Sci U S A. 9;112(23):7267-72. (2015). ...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...Most antibiotics target one of three essential processes: cell wall synthesis, DNA replication, or protein...

...CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. USA 112(10) (2015): 3002-7. PubMed... 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...Filonov, Gregory S., et al. “In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression...Aptamer-based assay for prion diseases diagnostic”. Procedia Environmental Sciences. Volume 12, Part B, 2012...

...expression and high cell-type specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type ...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression...

...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and... higher multiple circular DNA forms in bacteria. Proc Natl Acad Sci U S A. 1968 Dec;61(4):1406-13. doi... 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...

...mitigation with cross-over insensitive ATG-out vectors. Proc Natl Acad Sci USA 116:27001–27010. https://doi.org...one of lox2272 sites will now be sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This ...flank the transgenes (non-homologous pairs) (Figure 2.). They found that almost all off-target expression...exercise caution when using them.    Figure 2. Schematic diagram showing the mechanism of spontaneous... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLoS ONE 8:e76310... Pseudotyped with Viral Capsids from Serotypes 1, 2, and 5 Display Differential Efficiency and Cell Tropism...

...complete degradation of proteins in mammalian cells. Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):E3350-7....required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...

...Recruitment of CRISPR-Cas systems by Tn7-like transposons. Proc Natl Acad Sci USA 114:E7358–E7366 . https://doi....et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...genomic target site bound by QCascade (Fig. 1B). The process also creates a 5-bp target-site duplication (TSD...

...systematic studies of membrane protein interactions. Proc Natl Acad Sci U S A. 2004; 101(33):12242–12247. ...retain their individual activities when separated (2), and that DBD and TAs from different systems could... Enzymol. 1983;100:293-308. PubMed PMID: 6312261. 2. Keegan, L., Gill, G., and Ptashne, M. Separation ...

...genomics of biotechnologically important yeasts. Proc. Natl. Acad. Sci., 113, 9882-9887. DOI: 10.1073/...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...

... for real-time multicolor luminescence imaging.” Proc Natl Acad Sci U S A 112(14) (2015): 4352-6. PMID...improve the brightness of previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! ...activation, you can’t use fluorescence to monitor processes directly affected by light. Long-term light exposure...

...direct reprogramming by transformation-deficient Myc. Proc Natl Acad Sci U S A, 2010. 107(32): p. 14152-7. ...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...PMID: 27882265. PubMed Central PMCID: PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced ... to the process of collecting fibroblasts, which require a more invasive skin biopsy procedure. This becomes...measures, thereby increasing the costs of the procedure. Episomal reprogramming is an ideal and cost-effective...

...neurons with vesicular stomatitis virus vectors. Proc Natl Acad Sci USA 108, 15414–15419. PubMed PMID:...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...

...507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112... the genome of your favorite cells. Ma et al., Proc Natl Acad Sci U S A. 2015 Mar 10;112(10):3002-7....precision in a native context. Chatterjee A et al. Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11803-8... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...

...immunodeficiency virus type 1 preintegration complexes.” Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 6580–6584. PubMed...PubMed PMID: 8491198. PubMed Central PMCID: PMC413431. 2. Bukrinsky MI, et al. “Active nuclear import of human...

...immunohistochemical reagents. Yamagata M, Sanes JR Proc Natl Acad Sci U S A. 2018 Feb 13. pii: 1722491115...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus...would become busy and potentially detract from the process being depicted, we can’t forget that those other...plasmids through Addgene, don’t hesitate to start the process! Addgene depositors who study chromatin The PIs...

...with the endogenous tRNA-processing system. Xie K, Minkenberg B, Yang Y. Proc Natl Acad Sci U S A. 2015...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...conserved cellular machinery to process gRNAs. Hijacking tRNA processing to make gRNAs In a recently published...in rice plants. Yang was intrigued by the tRNA processing machinery present in almost every living organism...gRNAs with any 5’ leading nucleotide could be processed, a distinct advantage over other methods that ...is its universality; robust and efficient tRNA processing is common to prokaryotes and eukaryotes. Though...

...protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991;88(20):8972-6. Moks T,...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ... traditional, QC process. While this has worked well for the early sets of SARS-CoV-2 materials, it will...Bioworks to synthesize and share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...plasmid (packaging plasmid). Viral production then proceeds by transfection of all three plasmids into a producer...

...neurons in the adult dentate gyrus and olfactory bulb. Proc Natl Acad Sci U S A. 2013 Mar 19;110(12):E1152-61... 2008 Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, ...foundational later on when we studied biological processes, like how our stem cells are housed in specific...G-deleted RABV can divide and replicate (since these processes are mediated by other viral proteins), but the...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,...cells, wait 2–5 days and then collect the supernatant and/or producer cells for further processing. What component...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...over from the production process. For more details on the qPCR titering process, refer to the Addgene AAV...

...severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus may acquire mutations to escape ... SARS-CoV-2 infects host cells through binding the human angiotensin converting enzyme 2 (ACE2) cell-surface...Pooled library to study antibody escape in SARS-CoV-2 Recombinant antibodies for aminoacyl tRNA synthetases...viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple...viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing. From Starr... binding ACE2 is the target of most anti-SARS-CoV-2 antibodies. In the library, each plasmid encodes the...and a barcode, and is designed so that the SARS-CoV-2 spike RBD is displayed on the yeast cell’s surface...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...details. Our set of constructs comprises all SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. ...contaminants that have been used during the purification procedure. Good indicators of DNA purity are the absorbance...transfection grade DNA. Transfection is the process of inserting an external plasmid in mammalian cells...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to... an idea of the process, we’ll walk through an experiment designed to knock out exon 2 of a given gene...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...Gene targeting takes advantage of this natural process to replace a targeted genetic locus with homologous...

...determined by both codon bias and folding energy. Proc Natl Acad Sci USA 107:3645–3650 https://doi.org/...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...

...Biol. 2015. PubMed PMID: 25895059 Barnea, et al. Proc Natl Acad Sci U S A. 2008. PubMed PMID: 18165312...Science. 2013. PubMed PMID: 24136966 Nyerges, et al. Proc Natl Acad Sci USA. 2016. PubMed PMID: 26884157 ...generating protein chains or clusters. Veggiani, et al. Proc Natl Acad Sci U S A. 2016. PubMed PMID: 26787909...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally...

... 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...like Csy4, or the inclusion of processing signals, such at tRNAs, to process the array into functional crRNAs...Cpf1’s ability to process its own pre-crRNA arrays simplifies the crRNA cloning process. For cloning, Zetsche...

... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...that has defined our understanding of biological processes and developments. Model organisms often feature...history and characterization. Many proteins and processes naturally found in E. coli have been manipulated.... coli can be used as a model to relate these processes in more pathogenic bacteria (Blount, 2015). Rattus...McCusker and Gardiner, 2011). As many biological processes and signaling pathways that regulate regeneration...humans. Thus, understanding their life cycle and processes with their regenerative abilities is expected ...the most significant and fundamental biological processes. Many of their genetics and developmental patterns...

...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...

...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...and I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn...

.... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...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...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

...and Amy tell us a little about themselves. Chapter 2: What is Science in the News (SITN)? - SITN is a graduate...The SITN Editorial Process -Vini discusses the importance of the editorial process and peer-to-peer feedback...