... Depositor Collections MDH CUREs Community Malate Dehydrogenase CUREs Community...Research Experiences (CUREs) (Link opens in a new window) The Malate Dehydrogenase CUREs Community (MCC) (...hub of educators teaching MDH CUREs, and opportunities to mentor future MCC members. Find a regional hub...begin a sustainable MDH-related CURE focusing on protein expression, structure and function, kinetics and ...Resources MDH CUREs Community (Link opens in a new window) — Learn how to get started with a CURE, explore ...experience (CURE) projects. Depositor...Course-based Undergraduate Research Experiences (CUREs) centered on a single enzyme, malate dehydrogenase...

...downstream targets. Rajalingam K, Schreck R, Rapp UR, Albert S. Biochim Biophys Acta. 2007 Aug;1773(8)...

...addition of a structured motif to enhance pegRNA stability PEmax - optimized PE architecture; increased ...synthesized (Figure 8A), then cloned in a pooled format into lentiviral transfer vectors (Figure 8B). Libraries...Berkeley. Plan Your CRISPR Experiment Resources Figures All figures were created using BioRender.com (Link opens..., N., Ishitani, R., Zhang, F., & Nureki, O. (2014). Crystal Structure of Cas9 in Complex with Guide RNA...vivo genome editing using Staphylococcus aureus Cas9. Nature , 520 (7546), 186–191. PMID: 25830891 Rauch...became the most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism Engineered...gRNA left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will...

...isolated and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is...Antibodies can also be produced in cell cultures using hybridomas, cell cultures created by fusing B cells with...form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for binding the...Monoclonal antibodies are generated from cultured B cells, where each culture should produce the same antibody...Diabodies can be used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared... a signaling molecule. Figure 4: A direct sandwich ELISA, which uses a capture antibody and a primary ...In a sandwich ELISA, a capture antibody, bound to a surface, is used to capture the protein of interest...

...Featured article from Journal of Visualized Experiments (JoVE) about genomic deletions in mammalian cell...cell lines. Protocol For referenced Tables and Figures, please access the manuscript PDF posted at the...cleavage away from target sites (off-target effects). Ensure that the guide sequences consist of a 20-mer (“... or PAM) at the genomic recognition site. NOTE: Figure 1 describes possible deletion strategies for genes...nonsense mediated decay of the mRNA transcript ( Figure 1B ). Use the example guides for the intended deletion...deletion of Pim1 in mouse ( Mus musculus ; Table 1 , Figure 2A ). NOTE: In this example, sgRNA-A’s protospacer...sequence and PAM fall on the top (Watson) strand ( Figure 2A ). However, DSB will occur independent of orientation...

...Quantitative measurements of membrane charges A FRET sensor enables quantitative measurements of membrane...highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide...Aug 13;11(1):16519. Takeharu Nagai Temperature Fluorescent temperature indicator B-gTEMP Intracellular Heat...Campbell Voltage Multicoloured voltage reporters (QuasAr2) Bright and fast multicoloured voltage reporters...of plasmids encoding fluorescent biosensors to measure biomolecules or genes via FRET or other assays....enable investigation of a signaling pathway or measurement of a biomolecule while largely preserving spatial...fluorescent proteins for imaging neuronal activity. Nature. 2013 Jul 18;499(7458):295-300. Douglas Kim , GENIE...

...October 2013, Vol. 20 No. 10, pp.1296-304 Kogure et al. : Nature Biotechnology, May 2006, Vol. 24 No. 5,... et al. : Nature Methods, September 2007, Vol. 4 No. 9, pp. 741-6 Shemiakina et al. : Nature Communications...14003 Chu et al. : Nature Methods, May 2014 Vol. 11 No. 5, pp.572-8 Yu et al. : Nature Communications, May...for fluorescence to obtain half-maximal value. Structure describes the monomeric or oligomeric state of... many of these proteins, see: Cranfill et al. , Nature Methods 2016 Jul;13(7):557-62. FPbase : A database... (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Sirius 355 424 4 3.0 Prone to dimerization... (nm) Emission (nm) Brightness pKa Maturation Structure Plasmids Aquamarine 420 474 23 3.3 Prone to dimerization...

...DNA and cleaves it to destroy the invader ( Figure 1 ). Figure 1: An overview of the endogenous Type II ...vivo genome editing using Staphylococcus aureus Cas9. Nature . 520(7546):186-91. PMID: 25830891 Savic ... phages but also to destroy the phages upon re-exposure. More specifically, specialized Cas proteins snip...are grouped into two large classes based on the structure of the effector complex that cleaves the genomic... immune system seems like a winning strategy to ensure prokaryotic survival, but it is not invincible.... CRISPR-based genome editing in mammalian cell culture. Five years later, PubMed lists more than 6,300... loci with unprecedented speed and specificity. Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9...

...Anti-CASPR/Neurexin IV [K65A/30R] CASPR/Neurexin IV Rat Mouse IgG2a 219449 Anti-CASPR/Neurexin IV [K65A/...tissue culture cells (e.g., HEK293, COS, CHO), from which the R-mAbs are secreted into the culture media...IgG2a 114507 Anti-Neuregulin-HBD (Heparin binding domain, Type I/II) [N120A/9.1R] Neuregulin-HBD (Heparin ...Pan-SAPAP Rat Mouse IgG2a 114530 Anti-Neurexin-1-Beta [N170A/1.1R] Neurexin-1-Beta Human Mouse IgG2a 114533 ...Rat Mouse IgG2a 128625 Anti-CASPR/Neurexin IV [K66/38R] CASPR/Neurexin IV Rat Mouse IgG2a 128626 Anti-Nav1.1...Rat Mouse IgG2a 177445 Anti-CASPR/Neurexin IV [K65/35R] CASPR/Neurexin IV Rat Mouse IgG2a 177446 Anti-CASPR2...IgG2a 182103 Anti-Neuregulin-CRD (Cysteine-rich domain, Type III) [N126B/31R] Neuregulin-CRD (Cysteine-rich...

...Nishimasu H, Nureki O, Zhang F. Nature . 2015 Jan 29;517(7536):583-8. doi: 10.1038/nature14136. Epub 2014... Sharp PA, Zhang F. Nature . 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed...Science 2013, Ran et al ., Cell 2013, Ran et al ., Nature Protocols 2013). For cloning information, please... trans-activating crRNA (tracrRNA; Hsu et al ., Nature Biotechnology 2013). We also have an older system...be co-transfected as PCR amplicons (Ran et al., Nature Protocols 2013). Individual human codon-optimized...single vector system uses a smaller Cas9 from S. aureus (SaCas9) that has been human codon-optimized, and...guide RNA (sgRNA) into one plasmid (Ran et al., Nature 2015). The dual vector system uses S. pyogenes ...

...targeted to the same structures as these well-characterized proteins. General Structures The cell contains...of general cellular organelles and structures. ID Plasmid Structure Targeting Gene/Signal Fluorescent ...targeted to various organelles or subcellular structures. You should fuse your protein of interest with...contains a variety of organelles and subcellular structures each with their own unique function. The nucleus...required for chemical reactions. Labeling these structures can be quite useful for identifying the localization...internal framework that assists in sustaining structure and function. The cytoskeleton is primarily made... filaments, and intermediate filaments. These structures give the cell its shape, organize cellular organelles...

... are coming closer to this DNA targeting ideal. Figure 1: (A) Representation of a zinc finger (ZF) nuclease...useful protein domains to a specific DNA sequence ( Figure 1A ). Scientists have identified a large number...single nucleotide the TAL effector will recognize: ( Figure 1B ) HD targets cytosine, NI targets adenenine,...is on par with ZF arrays, if not slightly lower. Figure 2: Simplified representation of the Voytas/Bogdanove...-31 repeats. Perhaps one of the most appealing features of the TAL effector arrays is the ease with which...into a variety of backbones in just a few steps ( Figure 2 ). The Bogdanove group also hosts web-based software...The site, which will be updated soon with more features, is already receiving hundreds of unique visitors...

...human fibroblasts to neurons. Nature. 2011 Jul 13. doi: 10.1038/nature10323. Crabtree Fibroblasts Motor ... Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency. Cell. 2015 Jul 16;162(...Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach. Stem...cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25. 126(4):...cells with unique epigenetic and gene expression signatures. Cell Res. 2011 Mar;21(3):518-29. Cheng piggyBac... fibroblasts to induced pluripotent stem cells. Nature. 2009 Apr 9;458(7239):766-70. Nagy Lentivirus Mouse...cardiomyocytes using calcium activity as a functional measure of success. Mol Cell Cardiol. 2013 Apr 13;60C:97...

...design (meaning the mature processed form of crRNA is fused chimerically to a mature tracrRNA), the spacer... is based on the Cas9 D10A nickase described in Figure 4 of the Cong, et. al, 2013 Science paper. For ...genome editing, the Zhang lab has published this Nature Protocol article . Should I add the PAM sequence... to select your genomic target, you need to make sure the NGG immediately follows your target on the genome...-allelic cells usually make up the majority in culture unless the targeting efficiency is very high. How...NGA', or 'NGC' (if it's within coding region make sure it's a silent mutation). Is a protospacer followed...the Cas9. Again, if it's within coding region make sure it's a silent mutation. When attempting to use the...

...preparations are added to cells in culture. Three to five days later, cell cultures are inspected for sterility...droplet digital PCR (ddPCR). Quality Control Addgene ensures high quality viral vectors by optimizing and standardizing...quantity of gram-negative bacterial endotoxin is ensured to be less than 5 endotoxin units per mL. The endotoxin...used to determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified...corresponding catalog item (see maps section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...staining and appear darker than full vector particles. Figure 3: Electron micrograph of AAV vector preparation...packaging system developed by the Trono lab . Cell culture medium (OptiPro SFM) containing lentivirus is first...

...which are shipped as bacterial stabs at room temperature. Individual plasmids can be ordered from each...Thibodeau-Beganny S, Sander JD, Voytas DF, Joung JK. Nature Protocols . 2009;4(10):1471-501. doi: 10.1038/nprot...described by Hochschild and colleagues ( Dove et al., Nature 1997 ) and later modified by Pabo and colleagues... & Yeh et al., PLoS ONE 2009 , Townsend et al., Nature 2009 , Zou et al., Cell Stem Cell 2009 ). Resources... practicing OPEN is available in the 2009 Joung Nature Protocols paper . How to Cite this Kit These plasmids...include Addgene in the Materials and Methods of your future publications. For your Materials and Methods section...

...the permissive temperature (32°C) to allow transposition, and the construct is then cured by growing at...at the non-permissive temperature (42°C). Transposition is efficient enough that selection is not required...fluorescent protein variant (CyPet) optimized for FRET measurements with a yellow fluorescent protein variant (YPet...fluorescent protein variant (YPet) optimized for FRET measurements with a cyan fluorescent protein variant (CyPet...conditions in vitro , or, if you want to determine its structure using X-Ray crystallography, you’ll likely need...learn many things about your protein’s function, structure, and interactions! Plasmid ID Promoter Epitope...can be controlled by small molecules, light, temperature, or even intracellular metabolite levels. Different...

...plants - Jan Lohmann. MoClo Plant Parts II and Infrastructure Kit for hierarchical assembly of plant multigene...expression in plants. You can search the table for features you are looking for, such as the promoter or cloning...Proteins The table below highlights plasmids that feature fluorescent or luminescent proteins for expression...expression in plants. You can search the table for features you are looking for, such as the promoter or fluorescent... new window) - Collection of illustrations and pictures of plants, roots, shoots, and inflorescences -...responsible innovation for improvement of sustainable agriculture and conservation. Medicinal Plant Genomics Resource...publications are created. Papers from the plant biology literature are discussed with one of the authors. The podcast...

...demonstrates enhanced transduction of cells in culture for ex-vivo conditioning. The AAV2(trpYF) serotype...AAV2(Y444F) When using the AAV2(Y444F) serotype in future publications, please acknowledge Arun Srivastava...AAV2(trpYF) When using the AAV2(trpYF) serotype in future publications, please acknowledge Arun Srivastava...4pMut)dHS When using the AAV2(4pMut)dHS serotype in future publications, please acknowledge Shannon Boye and...T-V) When using the AAV6(dbY-F+T-V) serotype in future publications, please acknowledge Arun Srivastava...27308302 AAV44.9 When using the AAV44.9 serotype in future publications, please acknowledge John Chiorini ...E531D) When using the AAV44.9(E531D) serotype in future publications, please acknowledge Shannon Boye and...

...SynBio Blog Genome Engineering A distinguishing feature of synthetic biology is the ease with which parts...Enzymes: SpeI , NotI , PstI Scar: TACTAGAG or TACTAG Features: 8 bp Scar; No In-Frame Fusions; Coding Prefix...Suffix Enzymes: Nhel , NotI , PstI Scar: GCTAGT Features: 6 bp scar encodes Ala-Ser; Allows for in-frame...CTCGAG Suffix Enzymes: BamHI , Xhol Scar: GGATCT Features: Scar encodes Gly-Ser in-frame with prefix start...Suffix Enzymes: SpeI , NotI , PstI Scar: ACTAGA Features: Modified RFC 10 with shortened prefix/suffix ...Enzymes: Agel , Spel , Notl, Pstl Scar: ACCGGC Features: Modified RFC 10 w/ start and stop codons; Creates...