...references are below. Pooled genome-wide human SAM library SAM can be combined with a human genome-wide library...please visit the Addgene SAM library page . Targeted gene activation using SAM There are two sets of 3 ...CRISPR Blog Posts Jump to: SpCas9 Plasmids GeCKO SAM AAV - In vivo Delivery AAV - Cas9 Mouse CRISPR ( ...information. Full references are below. Return to top Cas9 SAM transcriptional activation plasmids and screening...library CRISPR/Cas9 Synergistic Activation Mediator (SAM) is an engineered protein complex for the transcriptional...available for mammalian endogenous gene activation using SAM: Addgene plasmids #61422 - #61424 were designed for...sites for insertion of guide sequences Zhang Lab SAM Cloning Protocol 321.5 KB Return to top AAV - In vivo...

...70,290 SAM v1 - 3 plasmid system 1000000075 (Puromycin) Activation Mouse Zhang 3rd 3 69,716 SAM v2 - 2...3rd 1, 2, or 4 49,766 arrays Broad GPP kinome Brunello 75314, 75315 (1 plasmid) 75312, 75313 (2 plasmid... Library 141438 Knockout Human Yeo 3rd 10 12,472 SAM v1 - 3 plasmid system 1000000057 (Zeocin) 1000000074...GPP genome-wide Brunello 73179 (1 plasmid) 73178 (2 plasmid) Knockout Human Doench and Root 3rd 4 76,441...Broad GPP genome-wide Brie 73632 (1 plasmid) 73633 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 78,637...172651 (Set D) Knockout Human Doench and Root 2nd 2 40,710 Inzolia Human CRISPR/Cas12a Multiplex Knockout...3,052 Broad GPP kinome Brie 75317 (1 plasmid) 75316 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 2,852...

... has increased stability and light output Sanjiv Sam Gambhir 104587 pHIV-iRFP720-E2A-Luc Firefly EF1α ...fusing NanoLuc® and Venus fluorophore to Troponin C Ca 2+ binding domain. Nano-lanterns , Enhanced Nano-lanterns...transfection Feng Zhang 21471 pLenti PGK V5-LUC Neo (w623-2) Firefly PGK Lentiviral expression of firefly luciferase...

...interface upon thaw (Fig. 2). Before thawing, all the samples were at the control pH of 7.4 as indicated by ...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...affected the pH of our viral samples. Read on to learn how aqueous samples might be affected by dry ice...Measuring the pH of our samples: What we did To measure the pH of our samples, we used a solution of cell...when the samples were frozen. This was clear by the gradual change in pH visible at the sample interface...at -80°C. For samples that are shipped on dry ice, this can affect the pH of the sample, which may have...

...different activators exist, including the multi-plasmid SAM system. Select Expression System To use CRISPR, you...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing...efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the...

...domains used in SAM. This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and...activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase...re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all shown significant improvements ... p65). Performance When targeting single genes, SAM consistently shows the highest levels of gene activation...regulation (activating multiple genes at once), however, SAM exhibits activation levels comparable to other popular...activators while showing lower activation levels than SAM. One drawback of this method is its construction:...VP64 activator, but lower levels compared to the SAM system. Its activation levels are similar to that...

...CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered protein complex for the transcriptional...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...not been achieved.” That is, until now. Enter the SAM pooled library In a paper published in Nature in ...plasmid which expresses the activation helper protein. SAM can be combined with a human genome-wide library ...Nature paper reports, “Here we have shown that the SAM system is robust, specific, and can facilitate genome-scale...combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited a high degree of consistency...References & Further Reading: For more information about SAM, including a tool for getting optimized guide sequences...

...Dilute oligos 1:10 in ddH 2 O ( e.g., 1.0 μl annealed oligos + 9.0 μl ddH 2 O to yield a concentration...been included. Ensure there are 2 x 10 6 cells per CRISPR pair. Resuspend 2 x 10 6 cells in 100 μl of electroporation...), 50-100 ng gDNA, and H 2 O up to 20 μl. Use the primers designed in step 2 above. Conduct PCR for “non-deletion...Run samples on 2% agarose gel at 10 V/cm using 1x Tris-acetate-EDTA (TAE) buffer. Examine samples for .... Pick 2 - 3 colonies and inoculate into a mini-prep culture. Perform mini-prep for each sample and sequence...deletion. Run sample in thermocycler and run the following program: 65 °C for 6 min, 98 °C for 2 min to extract... gDNA from clones. Run sample in thermocycler: 65 °C for 6 min and 98 °C for 2 min to extract gDNA. Screen...

...fusion none 2, 5 Richie 121539 pAAV SYN1 HA-hM3D(Gq) hM3D(Gq) - Activation HA fusion none 2, 5 Richie 50467...none 2, 8 Roth 50476 pAAV-CaMKIIa-hM3D(Gq)-mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, ...(Gq) - Activation mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 44362 pAAV-hSyn-DIO-hM4D...(Gi) - Inhibition mCherry fusion Cre-dependent 1, 2, 5, 8, 9, rg*, PHPeB Roth 65417 pAAV-hSyn-dF-HA-KORD-IRES-mCitrine...mCherry hM3D(Gq) - Activation mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 50475 pAAV-hSyn-hM4D(Gi)-mCherry...mCherry hM4D(Gi) - Inhibition mCherry fusion none 1, 2, 5, 8, 9, rg* Roth 52536 rAAV-CAG::FLEX-rev:: hM4D-...mCherry hM4D(Gi) - Inhibition mCherry fusion none 1, 2, 5, 8, 9 Roth 75033 pAAV CD68-hM4D(Gi)-mCherry hM4D...

...of basal H 2 O 2 levels with peroxiredoxin-based probes Real-time monitoring of basal H 2 O 2 levels with...Belousov Hydrogen Peroxide (H 2 O 2 ) Cytosolic or mitochondrial sensor for H 2 O 2 oxidation (roGFP2-Orp1) ...peroxiredoxin-2-based probe. Nat Commun. 2018 Aug 7;9(1):3145. Hadley Sikes Hydrogen Peroxide (H 2 O 2 ) Monitoring...encoded Ca(2+) indicator with enhanced two-photon absorption. Neurophotonics. 2024 Apr;11(2):024207. doi... Red fluorescent calcium sensors (fRCaMP1/2, GECO1/2) The kinetic mechanisms of fast-decay red-fluorescent...intracellular Zn(2+) homeostasis. Nat Methods. 2009 Oct;6(10):737-40. Maarten Merkx Zinc eZinCh-2 Zn2+ FRET ... eZinCh-2: A Versatile, Genetically Encoded FRET Sensor for Cytosolic and Intraorganelle Zn(2+) Imaging...

...immunoglobulin heavy diversity 2-15 D2, IGHD215 IGHD2-2 immunoglobulin heavy diversity 2-2 IGHD22 IGHD2-21 immunoglobulin...heat shock 70kDa protein 2 HSP70-2, HSP70-3 HSPA4 heat shock 70kDa protein 4 APG-2, HS24/P52, MGC131852, ...variable 2-33 (non-functional) IGLV233, V1-9 IGLV2-8 immunoglobulin lambda variable 2-8 IGLV28, V1-2 IGLV3...beta 4 DEFB-2, DEFB102, DEFB2, HBD-2, SAP1 EDN1 endothelin 1 ET1, HDLCQ7 EDN2 endothelin 2 ET2, PPET2 ...receptor subfamily 2, group C, member 1 TR2 NR2C2 nuclear receptor subfamily 2, group C, member 2 TAK1, TR2R1...suppression of tumorigenicity 2 - STC1 stanniocalcin 1 STC STC2 stanniocalcin 2 STC-2, STCRP TAC1 tachykinin,...FLJ99988, FLT2, HBGFR, KAL2, N-SAM, OGD FGFR2 fibroblast growth factor receptor 2 BEK, BFR-1, CD332, CEK3, ...

...extraction protocol for more details.   Figure 2: An uncut plasmid is expected to form 3 bands: nicked...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors...stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of these cloning... easiest and clearest ways to determine if your sample has recombined. Choose a restriction enzyme that...

...111707 Mode #2 Library (NEDD4 WW2 domain) Pooled Library 111708 Mode #2 Library (NEDD4-2 WW2 domain) References...Pooled Library 111705 Mode #2 Library (14-3-3β) Pooled Library 111706 Mode #2 Library (14-3-3σ) Pooled Library...phosphorylation-dependent protein-peptide interactions (“mode #2” expression, Barber et al, Nat. Biotech. 2018). The...fluorescence-activated cell sorting (FACS). The mode #2 phosphosite library with four different phosphobinding... WW2 domain of NEDD4, and the WW2 domain of NEDD4-2; 111705-8 ) is available from Addgene. Screening kinases...iSPI_pSer_Subpool#1 Pooled Library 188527 iSPI_pSer_Subpool#2 Pooled Library 188528 iSPI_pSer_Subpool#3 Pooled Library...pos ctrl Plasmid 111883 pNAS1b split mCherry NEDD4-2 WW2 Plasmid 111882 pNAS1b split mCherry NEDD4 WW2 ...

...suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the standard...catalog item (see maps section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre... a new vial of early passage cells. Approximately 2 weeks post-thaw, cell culture supernatant is tested...of the viral capsid proteins VP1, VP2 and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample. Sterility Viral vector preparations are added ...rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the.... Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received...every sample can be tracked easily at each step in the storage process. After the original sample is barcoded...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is contained... on the sample type. It is critical for us to know which plate, rack, and freezer each sample is located...

... mutations in the gene methyl-CpG binding protein 2 ( MECP2 ). MECP2 Rett syndrome is an X-linked disorder...X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet . 23, 185–188. (Link opens in a new window... Cuddapah et al. 2014. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity... Specific mutations in methyl-CpG-binding protein 2 confer different severity in Rett syndrome. Neurology...males with mutations in Methyl-CpG binding protein 2. Am J Med Genet B Neuropsychiatr Genet . 180, 55–67...variability in symptoms even within patients with the same mutation, the location and type of MECP2 mutation...cell line repository (Link opens in a new window) Sampled - one of the world's largest biorepositories (Link...

...overview of the most common methods. BioBrick BioBrick-2 BglBrick Silver Standard Freiburg Standard BioBrick...visit iGEM: BioBrick (RFC 10) Back to Top BioBrick BB-2 (RFC 12) Prefix: GAATTC GCGGCCGC T ACTAGT G Prefix...XbaI, SapI For more info, visit iGEM: BioBrick BB-2 (RFC 12) Back to Top BglBrick – Berkeley Standard ...assembly, even as more parts are added. Therefore, the same reaction which ligates a promoter into an empty ...

...strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected...realistic with the advent of CRISPR. Using CRISPR/Cas9 SAM to "shock and kill" To “shock and kill” the HIV-...the CRISPR/Cas9 Synergistic Activation Mediators (SAM) system available from Addgene. This system uses ...genome, which acts as a promoter, and found that SAM targeting of the enhancer region near the NF-KB binding...latent microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated ...that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill...CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation...

...sites on the same chromosome. Most multiplex systems enable researchers to target anywhere from 2–7 genetic...protein, instead of a two-component system (Figure 9C) SAM activators - co-expression of dCas9-VP64 with a modified...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...Cas9 is included in the gRNA-containing plasmid, or 2-vector systems, in which Cas9 must be delivered separately...your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either co-infect with...the presence of infectious organisms (like SARS-CoV-2 ) and genetic mutations. Similar to Cas9 and Cas12...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...

...cells; function is unknown Monomer with a valency of 2 IgE Protects against parasites and is responsible ...such as anaphylactic shock Monomer with a valency of 2 IgG The most abundant isotype in blood, also found...secondary immune response Monomer with a valency of 2 IgG1; IgG2; IgG3; IgG4 IgM Produced during the primary... ranging from antibody fragments (shown in Figure 2) to completely unrelated molecules. Examples include...used for assembling protein nanostructures. Figure 2: An antibody (IgG, far left) compared to alternative...application Collect and lyse samples to make proteins available. Tissue samples may need additional processing...culture samples to extract proteins. Homogenize (tissues only) and lyse. Cross-link the samples with formaldehyde...

...transcribed to make the pre-CRISPR RNA (pre-crRNA). (2) The pre-crRNA is processed into individual crRNAs...2015. Class 1 (Multi-subunit effector complex) Class 2 (Single multi-domain effector) Type I (Cas3) Type ... with unprecedented speed and specificity. Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9/gRNA...regulation of transcription in eukaryotes. Cell . 154(2):442-51. PMID: 23849981 Ishino Y, Shinagawa H, Makino...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell . 163(3):759-71. PMID: 26422227...targeting multiple loci can be easily expressed in the same cell or organism. The ability to precisely target...

...phage display. J Immunol Methods. 2000 Aug 28;242(1-2):159-81. Carlos Barbas Vector system for expression...+J phagemid libraries. Immunol Lett. 2015 Oct;167(2):95-102. Joanna Bereta Sybody Generation Toolbox for...microscope, or detect when the protein is present in a sample--just to name a few. Traditional sources of antibodies...

... Testing Samples Small samples of control AAV vectors for trial purposes in serotypes AAV1, 2, 5, 8, 9...Service overview. Function Chemogenetics DREADDs, PSAMs Optogenetics Channelrhodopsins, Halorhodopsins, ...

...Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time... the transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action ...

...referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the...standard consisting of ECFP and EYFP connected by 2 GGSGGS repeats pET28CLY3 Peptide linker standard consisting...the donor and acceptor are both fused to the same protein (also referred to as intramolecular or unimolecular...

...Cre-ERT2 CAG Mammalian Heller 112622 pVHC_PGKneoLox2DTA.2 Venus, Cre-ERT2, targeting vector with MCS for homology...homology arms Mammalian Heller 112623 pEHC_PGKneoLox2DTA.2 Emerald, Cre-ERT2, targeting vector with MCS for homology... arms Mammalian Heller 112624 pmTHC_PGKneoLox2DTA.2 TFP, Cre-ERT2, targeting vector with MCS for homology...arms Mammalian Heller 112625 ptdTHC_PGKneoLox2DTA.2 tdTomato, Cre-ERT2, targeting vector with MCS for ...25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed . Ventura A, Meissner A, Jaenisch R, Jacks...) If the target sites are on the same DNA strand (in cis) in the same direction, the sequence between ...orientation. A) If the target sites (loxP) are on the same DNA strand in opposite directions, recombination...

...8661 p4455 FLAG-hPLIC-2 UBQLN2 Flag CMV ALS Peter Howley 8662 p4456 FLAG-hPLIC-2 NTF UBQLN2 Flag CMV ALS...196208 GB1-A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep...196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196211 GB1-A11(2-196, G175R...196212 GB1-A11(2-196, G189E)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ...TLS 1: hTLS.pBSKS(+) FUS T7 ALS David Ron 21828 TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C...ApoE Alzheimer's Sohail Tavazoie 51436 pGL3Basic-ME.2/ApoEpromoter APOE ApoE Alzheimer's Sohail Tavazoie...T7 ALS Nicolas Fawzi 127195 RP1B FUS 1-163 QQ4xSS #2 FUS His T7 ALS Nicolas Fawzi 127196 pJ411 FUS 1-163...

...for shipping stability by incubating at 37 °C for 2 weeks, freezing at -80 °C, thawing on ice, and then...total protein content of the sample is determined. Only antibody samples with >90% purity are distributed...using a NanoDrop spectrophotometer. If needed, the sample is adjusted by diluting or concentrating to a final...Coomassie staining. For Coomassie staining, the sample is separated via a denaturing SDS-PAGE gel alongside...

...plasmid Bullock and Port 49409 pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...

...library substantially identified more hits than the SAM library approach in a comparison study screening ...screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation Mediator... PMID: 25184501. PubMed Central PMCID: MC4262738. 2. Doench, John G., et al. "Optimized sgRNA design to... in the depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello even with only...

...Gordon Fishell 83899-AAV1 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAV9 pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...Gordon Fishell 83899-AAVrg pAAV-mDlx-GCaMP6f-Fishell-2 GCaMP6f expression in forebrain GABA-ergic interneurons...eYFP Viviana Gradinaru 119741-AAV5 AAV SYN flex PSAM4 GlyR IRES EGFP Chemogenetics Cre-dependent expression...plasmid Scott Sternson 119741-AAV9 AAV SYN flex PSAM4 GlyR IRES EGFP Chemogenetics Cre-dependent expression...expression plasmid Scott Sternson 119742-AAV5 AAV SYN PSAM4 GlyR IRES EGFP Chemogenetics expression plasmid ...plasmid Scott Sternson 119744-AAV5 AAV CAMKII PSAM4 GlyR IRES EGFP Chemogenetics expression plasmid Scott Sternson...

...Information Featured Collections COVID-19 SARS-COV-2, ACE2, and CRISPR tools Fluorescent Proteins from ...allowed to use for distribution. Please see the Sample Agreements on our Technology Transfer Information...

...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...BclI-SwaI none S. pyogenes URA3 Wyrick pCRISPomyces-2 61737 Bacteria BbsI yes, cut S. pyogenes Apramycin... S. pyogenes Zhang gRNA Cloning Vector Bbs I ver. 2 85586 Mammalian BbsI None S. pyogenes H Fujii pX601...protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the...75026 Worm R07E5.16 (U6) none S. pyogenes Boxem lentiSAMv2 75112 Mammalian/Lentiviral U6 yes, activate S...

...transient mammalian expression 101126 pACP-tag (m)- 2 Vector ACP/MCP-tag Empty backbone for stable and transient...permitting selective simultaneous labeling in a sample. Please see the note below for information on substrates...

...include Cas9 fused to VP64, VP64 along with other SAM factors, and the Suntag array of factors, all targeted...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...recruitment of multiple activation domains to the same genetic locus via co-expression of an epitope tagged...

...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...for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the ...collects fluorescence. Like 2-photon microscopy, only a single focal plane of the sample is excited at one time...provide a 2-10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell...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...

...previously developed method to detect SARS-CoV-2 in the samples. First, RNA is extracted and subject to reverse... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...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...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

...sequence YFP Thorben Dammeyer 54520 mCherry-Peroxisomes-2 Peroxisome Peroximal Targeting Signal 1 (PTS1) mCherry...Centrin2 Centrioles (dependent on cell cycle) Centrin-2 EGFP Erich Nigg 41151 pEGFP Cep170 C-term Centrioles...can assess whether your protein is targeted to the same structures as these well-characterized proteins....

...loading control in the same lane, and the resulting fraction is reported. Figure 2: Normalization with...condition 1” than “condition 2.” By eye, the loading control appears about the same between the two conditions... than the analogous green “condition 2” points by the exact same value. For the two solid POI points, ... of your POI as a fraction of the total sample. Assumption #2: The loading control can be accurately quantified...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 ...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...

...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 ...Anti-SARS-CoV-2 Nucleocapsid Protein antibodies #211756 (left panel) or #211757 (middle panel). Samples were also...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...

...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

...arePharmacologically Selective Actuator Modules (PSAMs, pronounced SAMs). PSAMs are engineered α7 nicotinic acetylcholine...Figure 3: PSAM4-based ligand-gated ion channels, their effect, and outcome in neurons. Table 2. Descriptions...muscarinic receptor and has not been used in vivo . Figure 2: DREADDs, their ligands, and signaling properties.... Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 rM3D...Forbes SL, Liggett SB (2001). Modification of the beta 2-adrenergic receptor to engineer a receptor-effector...varenicline. This PSAM carries mutations at L131G, Q139L, and Y217F and is termed PSAM4 (or PSAM 4 ). Additional...

... step of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. The specific dilution series...series ranging from 1:2 to 1:32. Each individual step of the series is consistent at 1:2. When preparing a...of the series diluted by the same factor. For example, Figure 1 depicts a 2-fold dilution series. Each ...the unknown sample based on its measured response. An example of this is shown in Figure 2.   ...accuracy of the curve, avoid pipetting small volumes (< 2 µL) or volumes too large for a standard micropipette... Figure 2: The concentration of a serially diluted standard is plotted against its OD560 measurements...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...

...recruits transcriptional activators such as VP64, SAM, or SunTag to specific ncRNA loci to enhance or upregulate...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...insertions or deletions in the DNA sequence. This same approach is effective to target short ncRNAs, such...useful for targeting thousands of long ncRNAs at the same time (Liu et al. 2018). However, removing large ...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...hypodermis and gut cells of C. elegans causes the same phenotype. Activation of the Raf-MEK-ERK signaling...

... the same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of... when scientists talk about their precious RNA samples. RNA is inherently less stable than most macromolecules...CRISPR-Cas9 experiments – the “gRNA” – suffers from the same issues as your experimental RNA. To solve this problem...protein and can increase on-target efficiency. In the same vein, locked nucleic acids or bridged nucleic acids...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they have ... On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains ...stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction...cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e. tet-off transactivator...

...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...media will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 ...in a 10-100-fold increase in virus. Purification (2 days) - Purification is required if rAdV is to be ...